Nicotinamide adenine dinucleotide phosphate oxidase (nox) in experimental liver fibrosis: GKT137831 as a novel potential therapeutic agent.

Abstract

BACKGROUND & AIMS: NADPH oxidase (NOX) generates reactive oxygen species (ROS) in hepatic stellate cells (HSCs) during liver fibrosis. In response to fibrogenic agonists, such as angiotensin II (Ang II), the NOX1 components form an active complex including Rac1. Superoxide dismutase 1 (SOD1) interacts with the NOX-Rac1 complex to stimulate NOX activity. NOX4 is also induced in activated HSCs/myofibroblast by increased gene expression. Here, we investigate the role of an enhanced activity SOD1 G37R mutation (SODmu) and the effects of GKT137831, a dual NOX1/4 inhibitor, on HSCs and liver fibrosis. METHODS: To induce liver fibrosis, wild-type (WT) and SOD1mu mice were treated with carbon tetrachloride (CCl(4) ) or bile duct ligation (BDL). Then, to address the role of NOX-SOD1-mediated ROS production in HSC activation and liver fibrosis, mice were treated with a NOX1/4 inhibitor. Fibrosis and ROS generation was assessed by histology and measurement of TBARS and NOX related genes. Primary cultured HSCs isolated from WT, SODmu, and NOX1 knock-out (KO) mice were assessed for ROS production, Rac1 activity, and NOX gene expression. RESULTS: Liver fibrosis was increased in SOD1mu mice, and ROS production and Rac1 activity were increased in SOD1mu HSCs. The NOX1/4 inhibitor GKT137831 attenuated liver fibrosis and ROS production in both SOD1mu and WT mice as well as mRNA expression of fibrotic and NOX genes. Treatment with GKT137831 suppressed ROS production and NOX and fibrotic gene expression, but not Rac1 activity, in SOD1mut and WT HSCs. Both Ang II and TGFb upregulated NOX4, but AngII required NOX1. CONCLUSIONS: SOD1mu induces excessive NOX1 activation through Rac1 in HSCs, causing enhanced NOX4 upregulation, ROS generation, and liver fibrosis. Treatment targeting NOX1/4 may be a new therapy for liver fibrosis. (HEPATOLOGY 2011.).

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Nicotinamide Adenine Dinucleotide Phosphate Oxidase
in Experimental Liver Fibrosis: GKT137831 as a Novel
Potential Therapeutic Agent
Tomonori Aoyama,1,2Yong-Han Paik,3Sumio Watanabe,2Benoı ˆt Laleu,4Francesca Gaggini,4
Laetitia Fioraso-Cartier,4Sophie Molango,4Freddy Heitz,4C? edric Merlot,4C? edric Szyndralewiez,4
Patrick Page,4and David A. Brenner1
Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) generates reactive
oxygen species (ROS) in hepatic stellate cells (HSCs) during liver fibrosis. In response to
fibrogenic agonists, such as angiotensin II (Ang II), the NOX1 components form an active
complex, including Ras-related botulinum toxin substrate 1 (Rac1). Superoxide dismutase 1
(SOD1) interacts with the NOX-Rac1 complex to stimulate NOX activity. NOX4 is also
induced in activated HSCs/myofibroblast by increased gene expression. Here, we investigate
the role of an enhanced activity SOD1 G37R mutation (SODmu) and the effects of
GKT137831, a dual NOX1/4 inhibitor, on HSCs and liver fibrosis. To induce liver fibrosis,
wild-type (WT) and SOD1mu mice were treated with CCl4or bile duct ligation (BDL).
Then, to address the role of NOX-SOD1-mediated ROS production in HSC activation and
liver fibrosis, mice were treated with a NOX1/4 inhibitor. Fibrosis and ROS generation was
assessed by histology and measurement of thiobarbituric acid reactive substances and NOX-
related genes. Primary cultured HSCs isolated from WT, SODmu, and NOX1 knockout
(KO) mice were assessed for ROS production, Rac1 activity, and NOX gene expression. Liver
fibrosis was increased in SOD1mu mice, and ROS production and Rac1 activity were
increased in SOD1mu HSCs. The NOX1/4 inhibitor, GKT137831, attenuated liver fibrosis
and ROS production in both SOD1mu and WTmice as well as messenger RNA expression
of fibrotic and NOX genes. Treatment with GKT137831 suppressed ROS production and
NOX and fibrotic gene expression, but not Rac1 activity, in SOD1mut and WT HSCs. Both
Ang II and tumor growth factor beta up-regulated NOX4, but Ang II required NOX1.
Conclusions: SOD1mu induces excessive NOX1 activation through Rac1 in HSCs, causing
enhanced NOX4 up-regulation, ROS generation, and liver fibrosis. Treatment targeting
NOX1/4 may be a new therapy for liver fibrosis. (HEPATOLOGY 2012;56:2316-2327)
M
extracellular matrix (ECM) proteins, mainly collagen.1
Hepatic stellate cells (HSCs) play a key role in the
ost chronic liver diseases produce liver fibro-
sis, which results from the loss of hepato-
cytes combined with the accumulation of
response to hepatotoxic injury and are a major source
of ECM proteins.2Liver injury activates quiescent
HSCs to become myofibroblasts. Numerous studies
have now demonstrated that advanced liver fibrosis in
patientsandinexperimentalrodent modelsis
Abbreviations: Ab, antibody; Ang II, angiotensin II; a-SMA, alpha-smooth muscle actin; ALT, alanine aminotransferase; BDL, bile duct ligation; BM, bone
marrow; DCFDA, 2070-dichlorofluorescein diacetate; DMEM, Dulbecco’s modified Eagle’s medium; DPI, diphenyliodonium; ECM, extracellular matrix; GFP,
green fluorescent protein; FBS, fetal bovine serum; HSC, hepatic stellate cell; IF, immunofluorescent; IG, intragastric; 4-HNE, 4-hydroxynonenal; IHC,
immunohistochemical; IP, immunoprecipitation; Ki, inhibition constant; KO, knockout; LPO, lipid peroxidation; mAb, monoclonal antibody; MDA,
malondialdehyde; mRNA, messenger RNA; NADPH, nicotinamide adenine dinucleotide phosphate; NOX, NADPH oxidase; PBS, phosphate-buffered saline; PCR,
polymerase chain reaction; Rac1, Ras-related botulinum toxin substrate 1; ROS, reactive oxygen species; SEM, standard error of the mean; SOD1, superoxide
dismutase 1; SODmu, SOD1 G37R mutation; TBARS, thiobarbituric acid reactive substances; Tg, transgenic; TGF-b, transforming growth factor beta; TIMP-1,
tissue inhibitor of metalloproteinase 1; TNF-a, tumor necrosis factor alpha; XO, xanthine oxidase; WT, wild type.
From the1Department of Medicine, University of California San Diego, La Jolla, CA;2Department of Gastroenterology, Juntendo University School of Medicine,
Tokyo, Japan;3Department of Internal Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; and4GenKyoTex SA, Geneva,
Switzerland.
Received January 11, 2012; accepted June 13, 2012.
This work was supported by the American Liver Foundation (grant nos.: 1 R24 DK090962, 5 P50 AA011999, and 5 R01 GM041804).
2316

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reversible.3-5However, the only effective therapy to
treat hepatic fibrosis to date is to remove the causative
agent, so there is an unmet clinical need to develop
new, specific therapies for liver fibrosis.
Oxidative stress results from an inappropriate bal-
ance between the production and clearance of reactive
oxidative species (ROS) and leads to aberrant tissue
repair in the liver. Nicotinamide adenine dinucleotide
phosphate (NADPH) oxidase (NOX) is an enzyme
system that catalyzes the reduction of molecular oxy-
gen to superoxide. NOX in HSCs induces specific in-
tracellular signaling that results in activation.6The
NOX family consists of seven different members
(NOX1-5 and the dual oxidases, Duox1 and -2).7
Among the NOX family, both NOX1, NOX2 (also
named gp91phox), and NOX4 are expressed on HSCs
and may contribute to liver fibrosis.6,8Bone marrow
(BM) chimeric mice demonstrated that liver fibrosis
requires NOX2-generated ROS from both BM-derived
inflammatory cells and endogenous liver cells, includ-
ing HSCs, whereas NOX1 is required from only en-
dogenous liver cells.6Furthermore, NOX1 knockout
(NOX1KO) HSCs have less ROS generation than
NOX2KO HSCs.6Therefore, we suggest that NOX1
is more crucial than NOX2 in the generation of ROS
in HSCs. Upon stimulation with agonists, such as an-
giotensin II (Ang II), the cytosolic subunits, including
Rac-GTP, translocate to the membrane-bound cyto-
chrome complex to produce enzymatically active
NOX1 and NOX2.9On the other hand, NOX4 activ-
ity is regulated by increased expression of its protein,
including during myofibroblast/HSC activation.10-12
In particular, transforming growth factor beta (TGF-b)
signaling increases the protein expression and activity
of NOX through the increase in NOX4 gene tran-
scription, not by agonist-induced complex formation.7
Superoxide dismutase 1 (SOD1) interacts with Ras-
related botulinum toxin substrate 1 (Rac1) in the
active NOX complex to stimulate NOX activity.13
Mutations in SOD1, such as G93A and G37R, which
areassociated withfamilial
sclerosis,14increase NOX activity to produce increased
ROS in glial cells in the brain13and in other organs,
including the liver.15However, the interaction between
amyotrophic lateral
wild-type (WT) or mutant SOD1 with NOX in HSCs
and in liver fibrosis is unknown.
Because of this evidence incriminating NOX1 and
NOX4 in the pathogenesis of liver fibrosis, we aimed
toassess theeffectiveness
GKT137831, a NOX1/4 inhibitor, on the develop-
ment of liver fibrosis. Furthermore, We wanted to
investigate the role of SOD1 in NOX activity and liver
fibrosis. We hypothesized that mice with the SOD1
G37R mutation (SOD1mu) with increased catalytic
activity would have increased ROS generation and
increased liver fibrosis.
oftreatmentwith
Materials and Methods
Chemical
(dimethylamino)phenyl]-5-methyl-1H- pyrazolo[4,3-c]
pyridine-3,6(2H,5H)-dione was provided by Genkyo-
tex S.A. (Geneva, Switzerland).16GKT137831 is a
drug-like small molecule that was identified through
high-throughput screening, followed by medicinal
chemistry efforts involving hit-to-lead and lead optimi-
zation campaigns.17
Animal Models of Liver Fibrosis. Specific patho-
gen-free, WT C57BL/6J mice were purchased from
the Jackson Laboratory (Bar Harbor, ME). SOD1
G37R mutant mice in a C57BL/6 background were a
gift from Dr. Don Cleveland of the University of Cali-
fornia San Diego (San Diego, CA).18NOX1KO mice
in a C57BL/6 background were developed by K.H.
Krause, as previously described.19For the CCl4model
of liver fibrosis, 6-week-old male mice were injected
intraperitoneally with CCl4, which was diluted 1:3 in
corn oil (Sigma-Aldrich, St. Louis, MO), or with vehi-
cle (corn oil) at a dose of 0.5 lL/g of body weight
twice-weekly for a total of 12 injections. During the
last half of CCl4treatment, mice were treated with 60
mg/kg of the NOX1/4 inhibitor, GKT137831 (Gen-
KyoTex, Geneva, Switzerland) or vehicle by intragastric
(IG) injection daily. Mice were sacrificed 48 hours
after the last CCl4injection. For the bile duct ligation
(BDL) model, 6-week-old male mice were anesthe-
tized. After laparotomy, the common bile duct was
ligated twice and the abdomen was closed. The sham
GKT137831. 2-(2-chlorophenyl)-4-[3-
Address reprint requests to: David A. Brenner, M.D., Department of Medicine, University of California San Diego, 1318A Biomedical Sciences Building, 9500
Gilman Drive, La Jolla, CA 92093-0602. E-mail: dbrenner@ucsd.edu; fax: 858-822-0084.
Copyright V
View this article online at wileyonlinelibrary.com.
DOI 10.1002/hep.25938
Potential conflict of interest: B.L., F.G., L.F.-C., S.M., F.H., C.M., C.S., and P.P. are employees of GenKyoTex SA, which developed and provided the drug
(GKT137831) used in this study.
Additional Supporting Information may be found in the online version of this article.
C2012 by the American Association for the Study of Liver Diseases.
HEPATOLOGY, Vol. 56, No. 6, 2012AOYAMA ET AL.2317

Page 3

operation was performed similarly without BDL. From
11 days after the operation, mice were treated with 60
mg/kg of the NOX1/4 inhibitor, GKT137831, or ve-
hicle by daily IG lavage. Mice were sacrificed 21 days
after the operation.
Serum levels of alanine aminotransferase (ALT) were
measured with a commercial kit (Thermo Scientific,
Waltham, MA). Mice received humane care according
to the National Institutes of Health recommendations
outlined in the Guide for the care and Use of Labora-
tory Animals. All animal experiments were approved
by the institutional animal care and use committees
and performed at the University of California San
Diego.
HistologicalAnalysis. For
(IHC) analysis, liver specimens were fixed in 10% buf-
fered formalin and were incubated with monoclonal
antibody (mAb) against alpha-smooth muscle actin (a-
SMA; Sigma-Aldrich) with an M.O.M. kit (Vector
Laboratories, Inc., Burlingame, CA), or rat antimouse
F4/80 (eBioscience, Inc., San Diego, CA). For immu-
nofluorescent (IF) staining, frozen sections were incu-
bated with antibody (Ab) to SOD1 (The Binding Site
Group Ltd., Birmingham, UK), desmin (NeoMarkers,
Fremont, CA), or 4-hydroxynoneal (Alpha Diagnostic
International Inc., San Antonio, TX), and this was fol-
lowed by imaging with fluorescent microscopy.20
Western Blotting and Immunoprecipitation. The
preparation of whole cell protein extracts from frozen
livers, electrophoresis, and subsequent blotting were
performed as previously described.21Blottings were
with mouse Ab to a-SMA and b-actin (Sigma-
Aldrich), then visualized them with the enhanced
chemiluminescence light method (Thermo Scientific).
Immunoprecipitation (IP) of Rac1 was performed in a
LX-2 human HSC cell line. Dishes (15 cm) dishes of
LX-2 cells were pretreated with Ang II or phosphate-
buffered saline (PBS) for 30 minutes. Proteins were
extracted and incubated with mouse anti-Rac1 agarose
conjugate Ab (Millipore, Billerica, MA) for 4?C over-
night with rotating. Beads were washed five times with
lysis buffer. Pellets were resuspended in sodium do-
decyl sulfate polyacrylamide gel electrophoresis sample
buffer and incubated at 95?C for 5 minutes. Western
blotting was performed, as previously described, using
primary Abs to SOD1 (Binding Site) and Rac1 (Santa
Cruz Biotechnology, Inc., Santa Cruz, CA).
Isolation of Liver Cell Fractions. Liver cells of WT
mice were fractionated into four major cell populations
(hepatocytes, macrophages,
HSCs) with documentation of purity, as previously
described.6
immunohistochemical
endothelial cells, and
Quantitative Real-Time Polymerase Chain Reac-
tion. Total RNA was prepared from cells or frozen liv-
ers, reverse-transcribed, and quantitated by real-time
polymerasechainreaction
described.20PCR primer sequences are listed in the
Supporting information. The expression of respective
genes was normalized to 18S RNA as an internal
control.
Thiobarbituric Acid Reactive Substances Determi-
nation. Hepatic lipid peroxidation (LPO) was assessed
by measuring thiobarbituric acid reactive substances
(TBARS) formation.6Details are given in the Support-
ing Information.
HSC Isolation, Cell Culture, and Treatment. Mouse
HSCs were isolated using a two-step collagenase/pro-
nase perfusion of mouse livers, followed by 8.2%
Nycodenz (Accurate Chemical & Scientific Corpora-
tion, Westbury, NY) two-layer discontinuous density-
gradient centrifugation, as previously described.20After
isolation, HSCs were seeded on uncoated plastic tis-
sue-culture dishes and cultured in Dulbecco’s modified
Eagle’s medium (DMEM) (GIBCO BRL, Grand
Island, NY), supplemented with 10% fetal bovine se-
rum (FBS). HSCs isolated from WT, SOD1mu, or
NOX1KO mice were cultured in DMEM with 10%
FBS. After 48 hours of incubation, the medium was
changed into 1% FBS, and cells were then incubated
with 10?6M Ang II (Sigma-Aldrich) or vehicle (PBS)
with 20 lM of NOX1/4 inhibitor or vehicle (PBS) for
24 hours.
Measurement of Collagen-Driven GFP Expression
in HSCs. To measure collagen promoter activity,
HSCs (1 ? 105cell/well) were isolated from WT or
SOD1 mutant collagen promoter-driven green fluores-
cent protein (GFP) transgenic (Tg) (colI-GFP) mice
(pCol9GFP-HS4,5 transgene).22SOD1mu coll-GFP
mice were made by crossing WT coll-GFP mice with
SOD1mu mice. HSCs were incubated with 10?6M
Ang II (Sigma-Aldrich) or vehicle (PBS) with 20 lM
of NOX1/4 inhibitor or vehicle (PBS) for 24 hours.
The number of GFP-positive cells was determined by
the counting of GFP-positive cells in 10 randomly
chosen high-power fields.
Measurement of ROS Generation in HSCs. HSCs
were preincubated with the redox-sensitive dye, 2070-
dichlorofluorescein diacetate (DCFDA) (10 lM; Mo-
lecular Probes, Eugene, OR) for 20 minutes, then
stimulated with 10?6M Ang II (Sigma-Aldrich) or ve-
hicle (PBS) with 20 lM of NOX1/4 inhibitor or vehi-
cle (PBS). DCFDA fluorescence was measured with a
multiwall fluorescence scanner (FLUOstar OPTIMA;
BMG LABTECH, Ortenberg, Germany).6
(PCR), aspreviously
2318AOYAMA ET AL.HEPATOLOGY, December 2012

Page 4

Rac1 Activity Assay. Rac1 activity in HSCs was
determined with the Rac1 G-LISA activation assay kit
(Cytoskeleton, Inc., Denver, CO). Briefly, WT or
SOD1mu HSCs were treated by 10?6M Ang II
(Sigma-Aldrich) or vehicle (PBS) with 20 lM of
NOX1/4 inhibitor or vehicle (PBS) for 24 hours.
According to the manufacturer’s protocol, protein
lysate was extracted and Rac1 activity was determined
by luminescence intensity.
Statistical Analysis. Data are expressed as means 6
standard error of the mean (SEM). Statistical differen-
ces between means were determined using Student t
tests or analysis of variance on ranks, followed by a
post-hoc test (Student-Newman-Keuls’ all pairwise
comparison procedures), as appropriate. P values less
than 0.05 were considered statistically significant.
Results
SOD1 Expression Is Increased in HSCs in CCl4-
InducedFibrotic Liver. SOD1
(mRNA) levels were increased in the livers of WT
mice after CCl4-induced liver fibrosis (Fig. 1A). To
messenger RNA
investigate the cellular source of SOD1 in fibrotic
liver, we measured mRNA expression of SOD1 in he-
patocytes, macrophages/Kupffer cells, and HSCs iso-
lated from vehicle- or CCl4-treated mice. SOD1
expression was significantly increased in HSCs after
CCl4treatment, but not in hepatocytes or in macro-
phages (Fig. 1B). As detected by fluorescence micros-
copy, the number of SOD1- and desmin-positive
HSCs was markedly increased in CCl4-treated liver,
compared to vehicle control (Fig. 1C,D). These results
indicate that the up-regulation of SOD1 in HSCs is
related to the development of liver fibrosis.
GKT137831 Is a Novel, Specific Dual NOX1/4
Inhibitor. In an effort to discover new, selective mod-
ulators of Nox enzymes, we developed cell-free assays
using membranes prepared from cells heterologously
overexpressing a specific Nox enzyme isoform.23,24
GKT137831 (Fig. 2A) is a potent Nox4 inhibitor (in-
hibition constant [Ki] ¼120 6 30 nM) with an affin-
ity similar to the irreversible, unspecific flavoprotein
inhibitor, diphenyliodonium (DPI; Ki ¼ 70 6 10
nM) (Fig. 2B). As expected, DPI showed complete
nonselectivity, and the same potency was recorded on
Fig. 1. SOD1
enhanced in HSCs in the fibrotic
liver. Livers from WT mice were an-
alyzed after 12 injections of CCl4
or vehicle (n ¼ 5). (A) Hepatic
mRNA expression of SOD1 was
measured by quantitative real-time
PCR. (B) mRNA levels of SOD1 in
hepatocytes, macrophages,
HSCs isolated from vehicle- or
CCl4-treated WT mice detected by
quantitative real-time PCR (n ¼ 3).
(C) SOD1 expression (green) was
detected by fluorescence micros-
copy by containing with desmin
(red). (D) Percentage of SOD1-pos-
itive cells in desmin-positive cells.
expressionis
and
HEPATOLOGY, Vol. 56, No. 6, 2012 AOYAMA ET AL.2319

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all four Noxes probed (Fig. 2B). On the other hand,
GKT137831 had a better potency, both on human
Nox4 (Ki ¼140 6 40 nM) and human Nox1 (Ki
¼110 6 30 nM) and was found 15-fold less potent
on Nox2 (Ki ¼ 1,750 6 700 nM) and 3-fold less
potent on Nox5 (Ki ¼410 6 100 nM). Moreover,
GKT137831 did not significantly inhibit a highly spe-
cific NOX2-driven response (i.e., neutrophil oxidative
burst up to 100 uM), as measured by flow cytometry
in human whole blood. Also, the compound did not
show any immunosuppressive activity through poten-
tial inhibition of Nox2 when administered at 100 mg/
kg orally in an in vivo murine model of Staphylococcus
aureus killing (data not shown).
We demonstrated GKT137831 to be specific for
NADPH oxidases over other flavoprotein-containing
oxidasesand alsoexcluded
GKT137831 is a general ROS scavenger: GKT137831
was further tested in a xanthine oxidase (XO) assay
using similar ROS production methodology as in our
proprietary NOX assays and with the same readout.
Whereas DPI showed high affinity (Ki¼ 50 nM) con-
sistent with its nonspecific mechanism of action,
GKT137831 demonstrated no affinity for XO (Ki>
100 lM) (Fig. 2B,C) as well as the inability to scav-
enge superoxide (O2.?), the common endproduct of
Nox proteins and XO. To further demonstrate the
specificity of GKT137831 for Nox enzymes, our
candidate drug was subjected to an extensive in vitro
thepossibility that
off-target pharmacological profile on 170 different pro-
teins, including ROS-producing and redox-sensitive
enzymes, as well as representative proteins of well-
recognized drug target families, such as G-protein-
coupled receptors, kinases, ion channels, and other
enzymes.25GKT137831, when tested at 10 lM, did
not show any significant inhibition of any tested target
protein, demonstrating the excellent specificity of this
compound (see Supporting Table 2).
GKT137831 Prevents Liver Fibrosis in WT and
SOD1mu Mice. To investigate the role of SOD1 and
the effect of NOX1/4 inhibition on liver fibrosis, liver
fibrosis was induced in SOD1mu (with increased cata-
lytic activity) and WT mice by 12 consecutive CCl4
injections over a 6-week period. During the last half of
CCl4
injections,some mice
GKT137831 daily. CCl4-induced liver fibrosis was
more pronounced in SOD1mu, compared to WT,
mice. Liver fibrosis in both SOD1mu and WT mice
was attenuated byGKT137831
NOX1/4 inhibitor reduced the levels of hepatic colla-
gen deposition in CCl4-induced fibrosis in SOD1mu
and WT mice to the same low level, as assessed by
Sirius Red staining and its quantification (Fig. 3A,B).
Hepatic a-SMA expression, a marker for HSC activa-
tion, was enhanced in SOD1mu mice after CCl4injec-
tions, compared to WT mice, as assessed by IHC and
immunoblotting. The increased hepatic a-SMA expres-
sion was markedly decreased in SOD1mu mice treated
weretreatedwith
treatment.The
Fig. 2. Pharmacological profile of GKT137831, a dual Nox1/Nox4 inhibitor. (A) Chemical structure of the dual Nox1/Nox4 inhibitor,
GKT137831. (B) Inhibition of Nox-dependent ROS production by GKT137831: concentration-response curves of GKT137831 on membranes pre-
pared from cells specifically overexpressing hNox1 (^), hNox2 (D), hNox4 (o), hNox5 (!), and on XO (*). Michaelis constant (Km) of NADPH
for hNox1, hNox2, hNox4, and hNox5 was 70 6 10, 16 6 3, 120 6 20, and 70 6 10 mM, respectively, and Kmof xanthine for XO was 6 6
1 mM. Results are from one experiment performed in triplicate, representative of at least three experiments. Values are means 6 SEM. (C) Ki
value of GKT137831 and DPI on hNox1, hNox2, hNox4, hNox5, and XO.
2320AOYAMA ET AL.HEPATOLOGY, December 2012

Page 6

with GKT137831 to a level similar to that of WT
mice given the NOX1/4 inhibitor (Fig. 3C,D). The
mRNAs of fibrogenic markers, including collagen
a1(I), tissue inhibitor of metalloprotease 1 (TIMP-1),
and TGF-b were increased in SOD1mu mice to
higher levels than in WT mice after CCl4injections,
but treatment with GKT137831 reduced the induc-
tion of those genes to the same lower levels (Fig. 3E).
Similarly, BDL-induced hepatic fibrosis in both WT
and SOD1mu mice was decreased by treatment with
GK137831 (Supporting Fig. 1). Thus, both hepato-
toxin (CCl4)- (this study) and cholestasis (BDL)-
induced (this study and REFA) liver fibrosis is sup-
pressed by blocking NOX1 and NOX4.
GKT137831 Blocks Hepatic Inflammation in WT
and SOD1mu Mice. To investigate the role of SOD1
and the effect of NOX1/4 inhibition on liver inflam-
mation, macrophage infiltration and activation were
evaluated. After CCl4treatment, the expression of F4/
80 and CD68, a macrophage activation marker, was
significantly increased in SOD1mu versus WT livers, as
determined by IHC and quantitative real-time PCR.
However, NOX1/4 inhibition prevented macrophage
infiltration and activation to levels similar to those
observed in mice untreated with CCl4(Fig. 4A-C). He-
patic mRNA expression of tumor necrosis factor alpha
(TNF-a) was also increased in SOD1mu mice after
CCl4 treatment, but this increase was suppressed by
GKT137831 (Fig. 4D). Serum ALT levels were increased
in CCl4-treated SOD1mu mice, compared to CCl4-
treated WT mice, which was also reduced by NOX1/4
inhibition by GKT137831 (Fig. 4E). These results indi-
cate that increased liver inflammation and injury in
CCl4-treated SOD1mu and WT mice was inhibited to
similar lower levels by GKT137831 treatment.
Nox 1/4 Inhibition Decreases Hepatic LPO in
WT and SOD1mu Mice. To investigate ROS medi-
ated LPO, we measured the LPO products, 4-hydroxy-
nonenal (4-HNE) and malondialdehyde (MDA), as
indicators of oxidative stress in the liver. Hepatic
Fig. 3. Enhanced liver fibrosis
in SOD1mu mice is suppressed by
inhibitionof
GKT137831. Livers from WT or
SOD1mu mice were analyzed after
12 injections of CCl4or vehicle (n
¼ 5). In the last half period of
injections, some mice in each
strain were treated by NOX1/4 in-
hibitor daily. (A) Fibrillar collagen
deposition was evaluated by Sirius
Red staining (original magnifica-
tion: ?40), and (B) its quantifica-
tion is shown. The expression of a-
SMA in the liver was detected by
(C) IHC staining and (D) western
blotting(original
?100). (E) Hepatic expression of
collagen a1(I), TIMP-1, and TGF-b1
mRNA was measured by quantita-
tive real-time PCR. NI, NOX1/4 in-
hibitor. *P < 0.05.
NOX1/4 with
magnification:
HEPATOLOGY, Vol. 56, No. 6, 2012AOYAMA ET AL.2321

Page 7

4-HNE levels were increased in SOD1mu mice, com-
pared to WT mice, after CCl4 treatment, but this
increase was suppressed by inhibition of NOX1/4 (Fig.
5A). Measurement of MDA using TBARS showed
that increased hepatic MDA levels in SOD1mu mice
were suppressed by GKT137831 treatment after CCl4
injections (Fig. 5B). In agreement with liver fibrosis
and inflammation results, the levels of hepatic LPO in
CCl4-treated SOD1mu mice were decreased to the
same low level as WT mice by Nox 1/4 inhibition.
GKT137831 Inhibits Expression of Fibrogenic
and NOX Genes in HSCs. We used activation of pri-
mary cultures of HSCs as a model of activated myofibro-
blasts.26,27Primary HSCs were isolated from both WT
and SOD1mu mice. In quiescent HSCs, mRNA expres-
sion of collagen a1(I) and Acta2 are at the same low
level. mRNA levels in activated SOD1mu HSCs were
significantly greater than in activated WT HSCs (Fig.
5C). Incubating with GKT137831 reduced expression
levels of collagen a1(I) and Acta2 mRNA to the same
low levels in both activated SOD1mu and WT HSCs
(Fig. 5C). Interestingly, mRNA expression of NOX4
and, to a lesser extent, NOX1 was increased in activated
SOD1mu HSCs, compared to WT HSCs, and this
increase was also suppressed by GKT137831 (Fig. 5D).
GKT137831 Blocks Fibrogenic Response and ROS
Production Induced by Ang II in WT and SOD1mu
HSCs. Next, we assessed HSC activation by measuring
GFP fluorescence in quiescent HSCs purified from the
ColI-GFP reporter mouse, in which the collagen a1(I)
promoter/enhancer drives GFP. Ang II induced higher
GFP fluorescence in SOD1mu HSCs than in WT
HSCs, but this GFP enhancement was suppressed by
GKT137831 (Fig. 6A,B). To assess the effect of
SOD1mu on ROS generation, we measured the quan-
tity of ROS in DCFDA-loaded HSCs after Ang II
treatment. Ang II induced excessive ROS production
in SOD1mu HSCs,compared
GKT137831 suppressed ROS generation in SOD1mu
HSCs to the same levels as WT HSCs treated with
the NOX1/4 inhibitor (Fig. 6C).
To investigate whether SOD1 interacts with Rac1 in
response to Ang II, we performed coimmunoprecipita-
tion experiments for SOD1 and Rac1 in cell extracts
from the human HSC LX-2 cell line. SOD1 was
pulled down strongly by Rac1 after Ang II stimulation
in HSCs (Fig. 6D). Because the NOX-Rac complex
stimulates NOX activity, we assessed Rac1 activity in
HSCs after Ang II treatment. Rac1 activity increased
more in SOD1mu HSCs stimulated with Ang II than
inWTHSCs.As expected,
GKT137831 had no effect on Rac1 activity after Ang
II stimulation (Fig. 6E). Taken together, these results
indicate that the SOD1mut activates HSCs by forming
a complex with and activating Rac1. Treatment with
GKT137831 inhibits ROS generation, but does not
regulate Rac1 activity in both WT and SOD1mu
HSCs.
to WT HSCs.
treatmentwith
Fig. 4. Enhanced
SOD1mu mice is suppressed by NOX1/4 inhi-
bition. Livers from WT or SOD1mu mice were
analyzed after 12 injections of CCl4or vehicle
(n ¼ 5). In the last half period of injections,
some mice in each strain were treated by
NOX1/4 inhibitor daily. IHC for (A) F4/80 and
(B) its quantification are shown (original mag-
nification: ?100). (C and D) Hepatic mRNA
expression of CD68 and TNF-a was measured
by quantitative real-time PCR. (E) Serum ALT
levels were measured. NI, NOX1/4 inhibitor.
*P < 0.05.
liver inflammation in
2322AOYAMA ET AL.HEPATOLOGY, December 2012

Page 8

SOD1 G37R Mutation Enhances NOX4 Up-regu-
lation Through NOX1 in HSCs. To further investi-
gate the relationship between NOX1, NOX4, and
SOD1, HSCs were isolated from WT, SOD1mu, and
NOX1KO mice. In response to Ang II, induction of
NOX4mRNAexpression
NOX1KO HSCs, compared to WT HSCs (Fig. 7A).
These results indicate that NOX1 is required for
NOX4 up-regulation in HSCs stimulated with Ang II.
Ang II increased mRNA expression of both NOX1
and NOX4 in SOD1mut HSCs to a greater extent
than in WT HSCs. Treatment with GKT137831 sup-
pressed these increases to the same low levels in both
SOD1mut and WT HSCs (Fig. 7A). Similarly, Ang II
increased the mRNA expression of collagen a1(I) and
TIMP-1 more in SOD1mu HSCs, compared to WT
HSCs. The induction of these fibrogenic genes was
blocked in NOX1KO HSCs as well as by treatment of
WT and SOD1mu HSCs with the NOX1/4 inhibitor
(Fig. 7B). On the other hand, TGF-b induces NOX4
in HSCs independent of NOX1 (Fig. 7C).
was suppressedin
Discussion
Expression of NOX isoforms is increased in patients
with pulmonary,28renal,29and liver fibrosis.30Further-
more, NOX isoforms are induced and are required for
experimental murine models of pulmonary,10renal,31
and liver fibrosis.32HSCs require NOX for the fibro-
genic effects of Ang II,32leptin,33platelet-derived
growth factor,34TGF-b,10advanced glycation end-
products,35and phagocytosis.36Thus, NOX is a core
mediator37that is essential to convert an initial stimu-
lus to the development37of experimental and clinical
fibrosis in multiple organs. Our current study extends
our understanding of the role of NOX in hepatotoxic
and cholestatic liver fibrosis by demonstrating the fol-
lowing: (1) The catalytically active SOD1mut G37R
increases liver fibrosis in mice; (2) GKT137831, a
novel, first-in-class NOX 1/4 inhibitor, blocks liver fi-
brosis in SOD1mu and WT mice; (3) SOD1, Rac1,
and Nox1 interact to induce ROS and activate HSCs;
(4) Ang II induces Nox4 expression by Nox1 in
Fig. 5. Enhanced LPO and expression of
fibrogenic genes in
reduced by NOX1/4 inhibition. (A) Livers from
WT or SOD1mu mice were analyzed after 12
injections of CCl4or vehicle (n ¼ 5). In the
last half period of injections, some mice in
each strain were treated by NOX1/4 inhibitor
daily. Representative images of 4-HNE IF
staining (original magnification: ?100). (B)
Hepatic MDA levels were measured in livers
from (A) using TBARS assay. NI, NOX1/4 in-
hibitor. *P < 0.05. HSCs isolated from WT or
SOD1mu mice were incubated with NOX1/4
inhibitor (20 lM) or vehicle (n ¼ 3). mRNA
levels of (C) collagen a1(I) and Acta2 and
(D) NOX1 and NOX4 were measured by quan-
titative real-time PCR. qHSC, quiescent HSC;
aHSC, activated HSC; NI, NOX1/4 inhibitor.
*P < 0.05; #P < 0.05, compared to WT
aHSC. §P < 0.05, compared to SOD1mu
aHSC.
SOD1mu mice was
HEPATOLOGY, Vol. 56, No. 6, 2012AOYAMA ET AL.2323

Page 9

HSCs; (5) SOD1mu HSCs have increased fibrotic
gene expression, increased ROS production, and
increased Rac1 activity; and (6) GKT137831 blocks
the activation of SOD1mu and WT HSCs. Thus,
SOD1, NOX1, and NOX4 interact in HSCs to gener-
ate ROS and induce liver fibrosis.
SOD1, Rac1, and Nox1 Interact to Induce ROS
to Activate HSCs and Produce Liver Fibrosis. In
healthy cellular homeostasis, SOD1 converts superox-
ide to hydrogen peroxide to eliminate ROS.38Indeed,
treatment with an adenovirus vector encoding the
SOD1 gene improved ethanol-induced liver injury
through reducing free radical adducts in rats.39How-
ever, SOD1 also interacts with NOX, and certain
SOD1 mutations40induce the activation of NOX,
thereby causingadditional
tissues.13,15,41
ROS derived from NOX have an important role in
the development of liver fibrosis.6,32,42In the current
study, we demonstrate that SOD1 G37R mutation
worsens CCl4-induced liver fibrosis by increasing
NOX1/4 expression, Rac1 activity, and ROS genera-
tion in HSCs (Figs. 3-6). The mechanism for our ob-
ROSproduction in
servation is provided by the recent studies showing
that SOD1 stabilized Rac1, which is one of the cyto-
solic subunits interacting with NOX. Specific SOD1
mutations induce higher activation of NOX by main-
taining Rac1 in its active GTP-bound form, thereby
causingexcessiveROS
cells.13,43Consistent with these reports, our results
demonstrate that SOD1 interacts with Rac1, and
SOD1mu enhances Rac1 activity in HSCs treated
with Ang II (Fig. 6D,E). Thus, we propose that
SOD1/Rac1/NOX interaction is a core mediator in
HSC activation and fibrosis, including the fibrogenic
actions of Ang II on HSCs. Indeed, mRNA expression
of NOX1 and NOX4 was increased, accompanied by
enhanced fibrogenic responses in activated SOD1mu
HSCs, compared to activated WT HSCs (Fig. 5C,D).
Harraz et al. focused on NOX2 as a target of SOD1-
Rac1 component in glial cells.13Because NOX2 and
NOX1 share components, including Rac1 for their
activation,7and we showed that NOX1 is more im-
portant for ROS generation in HSCs than NOX2,6
targeting NOX1 is crucial for inhibiting excessive ROS
production in HSCs under fibrotic liver.
production andinjuring
Fig. 6. Increased fibrogenic response and ROS production stimulated by Ang II in SOD1mu HSCs are suppressed by NOX1/4 inhibition. HSCs
isolated from WT and SOD1mu colI-GFP Tg mice were stimulated by Ang II (10?6M), with NOX1/4 inhibitor (20 lM), or vehicle for 24 hours (n
¼ 3). (A) Representative photomicrographs of GFP-positive HSCs and (B) their quantification are shown (original magnification: ?200). *P <
0.05; #P < 0.05, compared to WT HSC stimulated by Ang II. §P < 0.05, compared to SOD1mu HSC stimulated by Ang II. (C) HSCs from WT
and SOD1mu mice were loaded with redox-sensitive dye (DCFDA; 10 lM) for 20 minutes. Cells were then washed twice and subsequently stimu-
lated by Ang II (10?6M) with NOX1/4 inhibitor (20 lM) or vehicle. Fluorescence signals were quantified continuously for 30 minutes using a
fluorometer (n ¼ 3). (D) Rac1 IP from cell extracts from the human HSC LX-2 cell line, followed by western blotting (WB) for SOD1 or Rac1. (E)
WT or SOD1mu HSCs were stimulated by Ang II with NOX1/4 inhibitor (20 lM) or vehicle for 24 hours. Rac1 activity was measured by enzyme-
linked immunosorbent assay (n ¼ 3). NI, NOX1/4 inhibitor. *P < 0.05.
2324 AOYAMA ET AL.HEPATOLOGY, December 2012

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Ang II Induces Nox4 Expression by Nox1 in
HSCs. NOX4 is regulated at the level of gene tran-
scription, not by the post-translational assembly of
components into a complex.7NOX4 is located down-
stream of TGF-b signaling and is an important mole-
cule in the activation of myofibroblasts.10-12Activation
of the TGF-b/Nox4 pathway has been shown to have
strong profibrotic activity in cardiac fibrosis,12kidney
fibrosis,13and lung fibrosis.11,19Inhibition of Nox4 in
activated myofibroblast either by knockdown with
short interfering RNA, or with the nonspecific irrevers-
ible NOX antagonist, DPI, prevented fibrosis in both
pulmonary11and kidney13fibrosis. In our study,
NOX4 mRNA levels were increased in activated and
Ang II–stimulated SOD1mu HSCs to a higher level
than in WT HSCs (Figs. 5D and 7A). These results
suggest that SOD1 regulates NOX4 induction. How-
ever, there are no studies reporting a direct interaction
between SOD1 and NOX4. Our study provides
insight into this relationship. First, because Ang II–
induced NOX4 mRNA expression was inhibited in
NOX1KO HSCs, compared to WT HSCs (Fig. 7A),
NOX1 induces NOX4 up-regulation in HSCs. Thus,
excessive activation of NOX1 by SOD1mu can lead to
increased NOX4 expression in HSCs (Figs. 5D and
7A). Second, previous reports demonstrated that Rac1
may regulate NOX4 in several cells.44-46
increased Rac1 activity in SOD1mu HSCs may induce
NOX4 up-regulation (Figs. 6E and 7C). Third,
increased phosphorylation of mothers against decapen-
taplegic homologs 2 and 3, which is downstream of
TGF-b signaling, was observed in the spinal cords of
SOD1mu mice,47demonstrating that an activating
SOD1 mutation leads to activation of TGF-b signal-
ing (Fig. 7C).
GKT137831, a Novel, First-in-Class NOX 1/4 In-
hibitor, Blocks Liver Fibrosis in SOD1mu and WT
Mice. The present study demonstrates the interaction
between SOD1, NOX1, and NOX4 to generate ROS
in HSCs and induce liver fibrosis. Building on the
work of us6,30,32,34,42and others,8,35,36,48this study
establishes a role for Nox1 and Nox4 in generating
oxidative damage to induce liver fibrosis. Treatment
with GKT137831, a novel, first-in-class, specific
Thus,
Fig. 7. Up-regulation of NOX4 by Ang II
and TGF-b. HSCs isolated from WT, SOD1mu,
and NOX1KO mice were stimulated by Ang II
(10?6M), TGF-b (10 ng), with NOX1/4 inhib-
itor (20 lM), or vehicle for 24 hours (n ¼
3). mRNA levels of (A) NOX1 and NOX4 and
(B) collagen a1(I) and TIMP-1 in HSCs after
Ang II stimulation. (C) mRNA levels of colla-
gen a1(I) and NOX4 after TGF-b stimulation.
NI, NOX1/4 inhibitor. *P < 0.05; #P <
0.05, compared to WT HSC stimulated by
Ang II or TGF-b. §P < 0.05, compared to
SOD1mu HSC stimulated by Ang II.
HEPATOLOGY, Vol. 56, No. 6, 2012 AOYAMA ET AL.2325

Page 11

Nox1/Nox4 inhibitor,17reverses fibrogenic response by
inhibiting ROS production and expression of fibro-
genic genes in both WT and SOD1mut HSCs. Most
important, GKT137831 blocks liver fibrosis and
down-regulates markers of oxidative stress, inflamma-
tion, and fibrosis in WT and SOD1mut mice. Taken
together, these results indicate that dual inhibition of
Nox1 and Nox4 might provide a unique opportunity
for the treatment of liver fibrosis and other fibrotic
diseases.
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