Targeting Nrf2 with the triterpenoid CDDO-
imidazolide attenuates cigarette smoke-induced
emphysema and cardiac dysfunction in mice
Thomas E. Sussana,1, Tirumalai Rangasamya,1,2, David J. Blakea,1, Deepti Malhotraa, Hazim El-Haddadb, Djahida Bedjac,
Melinda S. Yatesa, Ponvijay Kombairajua, Masayuki Yamamotod, Karen T. Libye, Michael B. Sporne,
Kathleen L. Gabrielsonc, Hunter C. Championb, Rubin M. Tuderf,3, Thomas W. Kenslera, and Shyam Biswala,f,4
aDepartment of Environmental Health Sciences, Bloomberg School of Public Health,bDivision of Cardiology,cDepartment of Molecular and Comparative
Pathobiology, andfDivision of Pulmonary and Critical Care Medicine, School of Medicine, Johns Hopkins University, Baltimore, MD 21205;dDepartment
of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Japan; andeDepartment of
Pharmacology, Dartmouth Medical School, Hanover, NH 03755
Edited by Gerald N. Wogan, Massachusetts Institute of Technology, Cambridge, MA, and approved November 19, 2008 (received for review May 5, 2008)
Chronic obstructive pulmonary disease (COPD), which comprises
emphysema and chronic bronchitis resulting from prolonged ex-
no effective treatment. Emphysema is also associated with pulmo-
nary hypertension, which can progress to right ventricular failure,
an important cause of morbidity and mortality among patients
with COPD. Nuclear erythroid 2 p45 related factor-2 (Nrf2) is a
redox-sensitive transcription factor that up-regulates a battery of
antioxidative genes and cytoprotective enzymes that constitute
the defense against oxidative stress. Recently, it has been shown
that patients with advanced COPD have a decline in expression of
the Nrf2 pathway in lungs, suggesting that loss of this antioxida-
tive protective response is a key factor in the pathophysiological
progression of emphysema. Furthermore, genetic disruption of
Nrf2 in mice causes early-onset and severe emphysema. The
present study evaluated whether the strategy of activation of Nrf2
and its downstream network of cytoprotective genes with a small
molecule would attenuate CS-induced oxidative stress and emphy-
sema. Nrf2?/?and Nrf2?/?mice were fed a diet containing the
potent Nrf2 activator, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-
28-oyl]imidazole (CDDO-Im), while being exposed to CS for 6
months. CDDO-Im significantly reduced lung oxidative stress, al-
veolar cell apoptosis, alveolar destruction, and pulmonary hyper-
tension in Nrf2?/?mice caused by chronic exposure to CS. This
protection from CS-induced emphysema depended on Nrf2, as
Nrf2?/?mice failed to show significant reduction in alveolar cell
apoptosis and alveolar destruction after treatment with CDDO-Im.
These results suggest that targeting the Nrf2 pathway during the
etiopathogenesis of emphysema may represent an important ap-
proach for prophylaxis against COPD.
chronic obstructive pulmonary disease ? oxidative stress
public health concern as it is currently the fourth-leading cause
of death in the United States (1). Emphysema, defined as
irreversible destruction of the alveoli, is associated with inflam-
mation in the airways and lung parenchyma. Emphysema is also
associated with pulmonary hypertension, which results from
destruction of the capillary network that is embedded in the
alveolar walls. Pulmonary hypertension leads to cor pulmonale,
which is an alteration of the structure and function of the right
ventricle (RV) that results from pulmonary hypertension. In
?3–5% of patients, cor pulmonale can progress to RV failure,
which contributes significantly to COPD-mediated mortality (2,
3). The primary risk factor for COPD is cigarette smoke (CS),
which accounts for ?80–90% of COPD cases worldwide.
Oxidative stress induced by CS plays a critical role in the
development of COPD. Markers of oxidative stress are elevated
hronic obstructive pulmonary disease (COPD), comprised
of emphysema and chronic bronchitis, represents a major
in both the lungs (4) and serum (5, 6) of COPD patients, and
enhanced oxidative stress leads to heightened inflammation and
alveolar cell apoptosis (7). A central hypothesis to explain the
promotes a protease/antiprotease imbalance (8), which leads to
enhanced elastolytic activity. The elevated elastin degradation
and reactive oxygen species (ROS) generated from the protease/
antiprotease imbalance in inflammatory cells may perpetuate a
positive feedback loop, which promotes further inflammation
and cell death. This loop may explain why ex-smokers have
persistent inflammation and alveolar destruction for several
years after quitting (9).
Although substantial progress has been made in understand-
ing many of the molecular mechanisms underlying COPD, this
knowledge has not translated into effective therapies. To date,
antioxidant therapies, such as N-acetylcysteine (NAC), have
failed to improve lung function or quality of life (10). Other
therapies that target inflammation, such as corticosteroids and
anti-TNF-? monoclonal therapy (infliximab), yield limited im-
provements on lung function (11–14). It is apparent that ap-
proaches reliant on stoichiometric scavenging of oxidants or
targeting the action of individual cytokines are not effective
there are genetic determinants of sensitivity to emphysema.
Recent studies from our laboratory and others demonstrate that
the transcription factor nuclear erythroid 2 p45 related factor-2
(Nrf2) is a key determinant of COPD susceptibility (15–17).
Nrf2 is a member of the basic leucine zipper (bZIP) family of
transcription factors that share a conserved cap ‘‘n’’ collar
domain (18, 19). Nrf2 functions as a critical mediator of an
adaptive response to counteract oxidative stress. Under basal
conditions, Nrf2 is tethered to its inhibitor Keap1, which facil-
itates its ubiquitination and proteolytic degradation in the
Author contributions: T.E.S., T.R., D.J.B., M.S.Y., K.T.L., M.B.S., K.L.G., R.M.T., T.W.K., and
S.B. designed research; T.E.S., T.R., D.J.B., D.M., H.E.-H., D.B., M.S.Y., P.K., and H.C.C.
performed research; M.Y., K.T.L., M.B.S., and R.M.T. contributed new reagents/analytic
tools; T.E.S., T.R., D.J.B., H.E.-H., D.B., and H.C.C. analyzed data; and T.E.S. wrote the paper.
Conflict of interest statement: M.B.S. is receiving grant support from Reata Pharmaceuticals.
This article is a PNAS Direct Submission.
1T.E.S., T.R., and D.J.B. contributed equally to this work.
Medical Center, Rochester, NY 14642.
3Present address: Department of Medicine, University of Colorado Health Sciences Center,
Denver, CO 80262.
4To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
January 6, 2009 ?
vol. 106 ?
cytoplasm. However, in the presence of electrophilic and/or
oxidative stresses, Nrf2 dissociates from Keap1 and translocates
to the nucleus, where it binds to other partner proteins and
activates the coordinate expression of a large number of anti-
oxidative and electrophile detoxification genes (20). Highlight-
ing the importance of this pathway in COPD, lung tissues and
alveolar macrophages from COPD patients exhibit decreased
Nrf2 pathway activation, compared with those from healthy
smokers (15–17). Furthermore, Nrf2?/?mice develop increased
alveolar destruction, and increased oxidative damage, apoptosis,
and inflammation, relative to Nrf2?/?mice, in response to chronic
CS (21, 22). This finding suggests that targeting the Nrf2 pathway
could have important clinical benefits for patients with COPD.
We have previously described that the synthetic triterpenoid
(CDDO-Im) acts as a potent activator of Nrf2 signaling in vitro
and in vivo (23–26). Furthermore, a close chemical congener of
CDDO-Im, namely CDDO-methyl ester, is currently in phase II
clinical trial for treatment of cancer (http://clinicaltrials.gov),
which suggests that CDDO-Im has potential to be a viable
therapy. In our present study, we tested whether targeting the
Nrf2 pathway with CDDO-Im, resulting in transcriptional in-
duction of antioxidative pathways, can attenuate pathological
lung damage and emphysema in mice after chronic CS exposure.
We have shown that CDDO-Im mitigates the development of
emphysema and its associated pathobiology; moreover, this
agent has beneficial systemic effects on pulmonary hypertension
and systolic and diastolic function of the RV.
CDDO-Im Reduces Alveolar Destruction After Exposure to CS in an
Nrf2-Dependent Manner. C57BL/6J Nrf2?/?and Nrf2?/?mice
were exposed to CS for 6 months, at which time detailed lung
morphological measurements were made. CS-induced airspace
enlargement was assessed by measuring the mean linear inter-
cept (MLI), which is an index of alveolar size, and alveolar
destruction was assessed by measuring surface–volume ratio
(S/V), which is an index of septal loss, using computer-assisted
stereologic measurements. MLI in Nrf2?/?mice exposed to CS
increased by 17.3% and S/V decreased by 13.7% when compared
with air-exposed controls (P ? 0.01), indicating that chronic CS
exposure significantly induced airspace enlargement (Table 1).
Furthermore, the CS-mediated alveolar destruction observed in
Nrf2?/?mice was significantly greater than in Nrf2?/?mice, after
CS exposure (P ? 0.05) (Table 1). One month of CS exposure
was not sufficient to significantly increase MLI in Nrf2?/?or
Nrf2?/?mice (data not shown). The aggregate of these data
demonstrates that Nrf2?/?mice have increased susceptibility to
CS-induced lung damage.
Earlier studies established that the Nrf2 activator CDDO-Im
exerted a pronounced pharmacodynamic action in the lungs of
mice, leading to enhanced Nrf2 transcriptional activity, after
oral administration (24). To determine whether CDDO-Im
protected against CS-mediated pulmonary pathological damage
and emphysema, mice were treated with CDDO-Im (60 or 90
mg/kg diet) throughout the 6-month CS exposure. There was no
significant alteration in weight gain with chronic feeding of
CDDO-Im (data not shown). Nrf2?/?mice that were treated
with CDDO-Im had significant improvement in both MLI and
S/V (P ? 0.05) when compared with vehicle-treated mice after
exposure to CS (Table 1). No dose response between 60 and 90
mg/kg was observed with regards to the lung protective effects;
however, the maximally effective dose could be ?60 mg/kg. In
Nrf2?/?mice, neither dose of CDDO-Im significantly improved
MLI or S/V. Therefore, the effects of CDDO-Im are mediated
in an Nrf2-dependent manner.
CDDO-Im Reduces Pulmonary Hypertension and RV Function After
Exposure to CS. To address whether CS impaired RV function in
tissue Doppler analysis, and pressure–volume loop catheteriza-
tion. CS exposure did not result in any significant alterations to
the left ventricle (data not shown). However, CS exposure
increased RV pressure and caused significant impairments to
RV diastolic and systolic functions (Table 2). In Nrf2?/?and
Nrf2?/?mice, CS caused a significant increase (45.5% and
42.5%, respectively) in RV end-systolic pressure (RVESP),
compared with air-exposed controls (Table 2). This increased
pressure was associated with a significant impairment in RV
ejection fraction (RVEF), demonstrating decreased RV con-
tractility (Table 2). CS exposure also caused prolongation of the
isovolumetric relaxation time (IVRT), demonstrating the pres-
ence of diastolic dysfunction (Table 2). Compared with Nrf2?/?
mice, Nrf2?/?mice had significantly decreased RVEF and
elongated IVRT, indicating that RV function in Nrf2?/?mice is
more adversely affected by CS than in Nrf2?/?mice.
CDDO-Im prevented the decrement of all 3 parameters of RV
heart function in Nrf2?/?mice (Table 2) to levels that were
comparable to those in air-exposed mice. CDDO-Im treatment
resulted in slight, but statistically insignificant, improvements in
heart function in Nrf2?/?mice. Therefore, CDDO-Im improved
heart function in a largely Nrf2-dependent manner, which
correlates with the reduction in airspace enlargement observed
in the lungs of mice treated with CDDO-Im.
Transcriptional Induction of Nrf2-Target Genes by CDDO-Im. Tran-
scriptional activity of Nrf2 is characterized by up-regulation of
expression of ?100 genes, many of which have antioxidative
functions (21). To determine whether CDDO-Im induced the
expression of Nrf2 target genes after chronic CS exposure, we
examined gene expression of several known Nrf2-dependent
Table 1. Effect of CDDO-Im on lung morphometry after chronic exposure to CS
MLI, ?mS/V (?1,000)
MiceCDDO-Im, mg/kg Air CS % IncreaseAirCS% Increase
0 39.4 ? 1.0
39.4 ? 2.0
39.8 ? 0.7
42.3 ? 0.7
42.5 ? 0.5
43.0 ? 0.3
46.2 ? 0.3*
41.4 ? 0.6†
42.8 ? 0.6†
52.2 ? 1.4*‡
52.8 ? 1.3*
50.7 ? 1.2*
50.1 ? 1.4
49.3 ? 1.3
49.3 ? 2.7
45.6 ? 1.2
45.4 ? 0.9
44.0 ? 1.4
43.3 ? 0.7*
48.2 ? 1.0†
46.4 ? 0.9†
38.7 ? 1.4*‡
38.9 ? 0.7*
38.2 ? 1.6*
N ? 4–6 per group. P ? 0.05 was considered significant by Student’s two-tailed t test.
*CS-exposed Nrf2?/?and Nrf2?/?mice exhibited significant airspace enlargement relative to air-exposed controls.
†Nrf2?/?mice treated with CDDO-Im exhibited significant improvement relative to Nrf2?/?without CDDO-Im after CS exposure.
‡Nrf2?/?mice exhibited significant airspace enlargement relative to Nrf2?/?after CS exposure.
Sussan et al.
January 6, 2009 ?
vol. 106 ?
no. 1 ?
genes in the lungs after 6 months of CS exposure. Lungs were
harvested 18 h after the final air/CS exposure. In general, Nrf2
target genes in CS-exposed mice were elevated, compared with
air-exposed mice (Fig. 1). CS-exposed Nrf2?/?mice treated with
CDDO-Im exhibited higher expression of several Nrf2 target
genes, compared with CS alone. As expected, CDDO-Im did not
induce Nrf2-dependent gene expression in Nrf2?/?mice. This
observation demonstrates that CDDO-Im enhances the Nrf2-
dependent antioxidative response in chronic CS-exposed mice.
We also examined gene expression 18 h after an acute (1 day)
CS exposure. The acute CS-exposed mice did not exhibit in-
creased Nrf2-target gene expression compared with air-exposed
mice (Fig. 1). However, among CS-exposed mice, mice treated
with CDDO-Im had elevated Nrf2-dependent gene expression.
Thus, CDDO-Im activates Nrf2-dependent responses after both
acute and chronic exposures to CS.
CDDO-Im Reduces CS-Induced Apoptosis and Oxidative Stress in the
Lungs. Emphysema has been linked with alveolar cell apoptosis
and enhanced oxidative stress (27). Therefore, we investigated
whether CDDO-Im-mediated induction of cytoprotective path-
ways led to decreased oxidative stress and apoptosis in Nrf2?/?
mice. Exposure to CS for 6 months led to an increase in the
number of TUNEL-positive cells in both Nrf2?/?and Nrf2?/?
mice. However, this increase was 6-fold greater in Nrf2?/?mice,
compared with Nrf2?/?mice (Fig. 2). One month of CS exposure
was not sufficient to increase TUNEL staining in either Nrf2?/?
or Nrf2?/?mice (data not shown). Furthermore, the increase in
apoptosis after 6 months of CS corresponded with an increase in
cell proliferation in Nrf2?/?but not in Nrf2?/?mice (Fig. S1). In
Nrf2?/?mice, treatment with CDDO-Im reduced the number of
alveolar apoptotic cells to levels observed in air-exposed mice,
which was associated with a significant decrease in cellular
apoptotic or proliferative cells in Nrf2?/?mice, after CS expo-
sure. Therefore, the apoptotic profile mirrored the effects of
CDDO-Im treatment on lung morphometry, suggesting that
apoptosis closely parallels CS-induced alveolar destruction.
We also assessed whether CDDO-Im reduced oxidative dam-
age after 6 months of CS exposure. To determine the extent of
oxidative damage, lungs were stained for the DNA adduct,
8-oxo-7,8-dihydro-2?-deoxyguanosine (8-OHdG). Although CS
caused increased 8-OHdG staining in both Nrf2?/?and Nrf2?/?
mice, the level of oxidative damage was significantly greater in
oxidative damage in Nrf2?/?, but not Nrf2?/?mice after chronic
CS exposure. One month of CS exposure led to only subtle
increases in 8-OHdG staining in Nrf2?/?mice (data not shown).
We also measured oxidative stress after acute exposure to CS.
Mice were exposed to CS for 1 day, and levels of the major
antioxidant tripeptide, glutathione, was measured in the lungs.
After exposure to CS, glutathione levels decreased significantly
in both Nrf2?/?and Nrf2?/?mice (Fig. S2A). CDDO-Im signif-
icantly elevated glutathione levels in both air- and CS-exposed
Nrf2?/?mice, compared with their respective controls (Fig.
S2A). In CS-exposed Nrf2?/?mice, CDDO-Im caused a slight
increase in glutathione concentration compared with untreated
mice. However, the glutathione concentration remained below
the level observed in Nrf2?/?air-exposed mice. These results
suggest that CDDO-Im induces a protective antioxidant re-
sponse that is absent in Nrf2?/?mice. Glutathione levels after 1
month of CS exposure were comparable to those observed after
1 day of CS exposure (Fig. S2B).
Our findings demonstrate that Nrf2?/?mice have increased
alveolar destruction after exposure to chronic CS. The enhanced
susceptibility of Nrf2?/?mice to CS is consistent with previous
studies in both humans and mice (15–17, 21, 22). The decreased
Nrf2 activity in the lungs and macrophages of COPD patients,
and the increased susceptibility of Nrf2?/?mice to CS-mediated
emphysema, led us to hypothesize that activation of Nrf2 would
attenuate the lung destruction caused by CS. We showed in this
study that transcriptional induction of Nrf2-regulated antioxi-
dative genes by the small-molecule activator, CDDO-Im, re-
duced alveolar destruction, lung apoptosis, and oxidative stress
imposed by CS exposure.
Cor pulmonale is a common complication of emphysema, and
it is closely associated with COPD-mediated mortality. In this
study, CDDO-Im reduced RV pressure and improved RV
contractility and relaxation. Thus, CDDO-Im can reduce alve-
olar destruction and improve a major cardiac determinant of
COPD-related mortality. Furthermore Nrf2?/?mice exhibited
significantly reduced RV function, compared with Nrf2?/?mice,
despite similar RV pressures. This result suggests that Nrf2 is a
determinant of RV function, and it is direct in vivo evidence
demonstrating that Nrf2 activity impacts cardiac physiology.
CDDO-Im is an exceptionally potent activator of the Nrf2
pathway, exhibiting effects at high picomolar to low nanomolar
concentrations in vitro (23, 26). However, at higher concentra-
tions, CDDO-Im has been shown to activate other signaling
pathways (25). Although CDDO-Im is a pleiotropic molecule
with multiple activities, our studies clearly demonstrate that, at
the doses used in this study, CDDO-Im primarily acted in an
Nrf2-dependent manner, because CDDO-Im did not signifi-
cantly reduce indicators of emphysema in Nrf2?/?mice after
chronic exposure to CS.
CS is a potent generator of reactive oxygen and nitrogen
species, which cause damage to nucleotides, proteins, and lipids.
This macromolecular damage, in turn, leads to elevated apopto-
sis and inflammation. In this study, we showed that CDDO-Im
significantly reduced oxidative stress and apoptosis in the lungs
of CS-exposed Nrf2?/?mice, but not in Nrf2?/?mice. However,
CDDO-Im did not reduce the number of inflammatory cells in
Table 2. Effect of CDDO-Im on end-systolic pressure, ejection fraction, and IVRT of the RV after chronic exposure to CS
Pressure, RVESP, mmHG Systolic function, RVEF, % Diastolic function, IVRT, msec
% Air CS
%Air, msec CS, msec
0 22.4 ? 0.9 (7)
25.2 ? 1.2 (6)
23.6 ? 0.5 (5)
24.7 ? 0.6 (6)
32.6 ? 1.3 (13)*
26.3 ? 0.8 (7)†
34.2 ? 1.4 (5)*
30.5 ? 2.5 (3)
67.7 ? 2.9 (7) 56.3 ? 2.9 (8)*
63.2 ? 1.2 (8) 66.2 ? 3.1 (7)†
63.5 ? 1.9 (5) 36.8 ? 3.8 (8)*‡
64.6 ? 1.3 (7) 44.4 ? 3.1 (3)
19.6 ? 2.3 (7) 26.0 ? 1.3 (6)*
21.9 ? 1.1 (7) 23.0 ? 1.9 (5)
20.5 ? 0.5 (6) 34.8 ? 1.4 (5)*‡
22.0 ? 0.9 (6) 27.3 ? 1.2 (3)
Number of mice per group is listed in parentheses. P ? 0.05 was considered significant by Student’s two-tailed t test.
*CS-exposed Nrf2?/?and Nrf2?/?mice exhibited significant cardiac impairment relative to air-exposed controls.
†CDDO-Im caused significant improvement in Nrf2?/?mice, relative to Nrf2?/?without CDDO-Im after CS exposure.
‡Nrf2?/?mice had a significant impairment relative to Nrf2?/?after CS exposure.
www.pnas.org?cgi?doi?10.1073?pnas.0804333106Sussan et al.
bronchoalveolar lavage fluid and lung parenchyma in CS-
that CDDO-Im can attenuate expression of neutrophilic cyto-
kines and chemokines after acute challenge with LPS (28) or
Con A (29).
In response to oxidative stress, Nrf2 coordinately up-regulates
the expression of a large cohort of antioxidative and xenobiotic
detoxication genes and activates enzymes involved in regenera-
tion of glutathione, thioredoxin, and NADPH. The success of
CDDO-Im in preventing the development of emphysema may be
caused by its activation of multiple Nrf2-dependent genes. Other
antioxidant and antiinflammatory therapies target single anti-
oxidative genes or cytokines, which may limit their effectiveness.
Reduced Nrf2 activity in patients with advanced COPD lends
support to the importance of an antioxidant-coordinated re-
sponse in the pathogenesis of CS-induced alveolar destruction
(15–17). Furthermore, although most current therapies target
inflammation, treatment with CDDO-Im did not reduce inflam-
mation, suggesting that cytoprotection of lung parenchyma from
oxidative stress is sufficient to protect against emphysema.
In this study, CDDO-Im was delivered concurrently with CS
exposure. This treatment strategy suggests that CDDO-Im could
have beneficial effects as a secondary prophylactic that could
delay or prevent progression of COPD. Our results provide
promise for future clinical trials that target the Nrf2 pathway in
active or passive smokers with COPD.
Materials and Methods
Animals and Treatments. Nrf2?/?and Nrf2?/?C57BL/6J mice were housed
under controlled conditions for temperature and humidity, using a 12-h
light/dark cycle. At 10 weeks of age, mice were exposed to CS for 5 h/day, 5
1-day group by gavage before air/CS exposure. Lungs from mice were harvested
by TaqMan. Solid circles represent Nrf2?/?mice, and open circles represent
was set to 1. Nrf2-target genes analyzed were: heme oxygenase 1 (Ho1), NAD-
(P)H:quinone oxidoreductase 1 (Nqo1), glutamate cysteine-ligase catalytic sub-
unit (Gclc), glutamate cysteine-ligase modifier subunit (Gclm), sulfiredoxin 1
(Srx1), and glucose-6-phosphate dehydrogenase (G6pd). n ? 3 per group.*, P ?
0.05 by Student’s 1-tailed t test.
Relative fold induction of Nrf2-target gene expression. Mice were
stained for TUNEL. (Magnification: 100?.) (B and C) Quantification of TUNEL-
positive cells (per 100 DAPI-positive cells) is shown for Nrf2?/?(B) and Nrf2?/?
(C) mice that received either CDDO-Im or control diet. For each mouse, a
minimum of 5 fields were captured. n ? 4 per group.*, P ? 0.05 by Student’s
2-tailed t test.
Treatment with CDDO-Im reduced CS-induced apoptosis in the lungs
Sussan et al.
January 6, 2009 ?
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no. 1 ?
and 2R4F reference cigarettes (University of Kentucky, Tobacco Research
Institute, Lexington). Chamber atmosphere was monitored for total sus-
pended particles and carbon monoxide, with concentrations of 90 mg/m3and
350 ppm, respectively. For alveolar morphometry, mice were fed with
For all subsequent experiments, mice were treated with the 90 mg/kg diet
dose. All experimental protocols were performed in accordance with the
Johns Hopkins University Animal Care and Use Committee.
(13.5 mg/kg body weight) or vehicle at ?48, ? 24, and 0 h before being
dietary level given for chronic exposure studies. CDDO-Im was synthesized as
described (30) and obtained from Reata Pharmaceuticals.
Lung Morphometry. Eighteen hours after the final CS exposure, lungs were
inflated with 0.6% agarose at a constant pressure of 25 cm H2O, as described
(31). Lungs were fixed for 24 h in 10% buffered formalin and embedded in
were captured at 100? magnification. MLI and S/V ratio were determined by
using a macro designed with MetaMorph software (Molecular Devices).
Heart Physiology. RVEF was determined in conscious mice by 2D echocardiogra-
the Simpson method using the apical 4-chamber view. IVRT was measured
was determined in anesthetized mice with an SPR-839 4 electrode pressure-
and positioned along the longitudinal axis to record chamber volume by
impedance and pressure by micromanometry.
TUNEL Assay. Apoptotic cells were quantified in the lung parenchymal tissue
with the TdT-FragEL DNA Fragmentation Detection Kit (Calbiochem) accord-
ing to the manufacturer’s instructions. Lung sections were stained with
Vectashield mounting medium for fluorescence (Vector Laboratories). The
number of apoptotic (TUNEL positive) cells was quantified by using the
Elements software package (Nikon Instruments). The software determined
the number of TUNEL-positive cells through intensity and size of 3,3?-
diaminobenzidine staining in each nucleus. Nuclei were identified by double
labeling with DAPI.
Oxidative Stress Markers. The occurrence of oxidative stress in the lung
sections was assessed with an anti-8-OHdG antibody (QED Biosciences), fol-
counted manually at 100? magnification.
For determination of glutathione concentration, lungs were harvested
M sucrose, 10 mM Tris?HCl, and 1 mM EDTA. Protein was precipitated by
adding sulfosalicylic acid to a final concentration of 6.5%, followed by incu-
bation on ice for 10 min and centrifugation at 2,000 ? g for 15 min. Total
intracellular glutathione was measured by using the glutathione reductase-
5,5?-dithiobis(2-nitrobenzoic acid) recycling assay at 412 nm. Total intracellu-
lar glutathione levels were determined by quantifying the intracellular glu-
tathione levels and dividing by the protein concentration. The data are
expressed as nmol glutathione per mg protein.
Gene Expression. Total RNA was isolated from lungs 18 h after the last CS
exposure, using TRIzol reagent (Invitrogen), and cDNA was generated by
using Multiscribe reverse transcriptase (Applied Biosystems). Gene expression
was measured using assays on demand probe sets (Applied Biosystems), and
reactions were analyzed by using the ABI 7000 Taqman system.
Inflammation. Inflammation was measured in lung tissues of chronically ex-
posed mice by immunohistochemical staining of macrophages, using the
Mac3 antibody (BD Biosciences as described by the manufacturer. Inflamma-
tion was measured in bronchoalveolar lavage fluid, as described (21).
Statistical Analyses. Student’s t test was used to determine statistical signifi-
cance between each group. Values are presented as means ? standard error.
ACKNOWLEDGMENTS. This work was supported by National Institutes of
Health Grant HL081205 (to S.B.), National Heart, Lung, and Blood Institute
Specialized Centers of Clinically Oriented Research Grant P50HL084945, Na-
tional Cancer Institute Grant CA78814 (to M.B.S.), and CA94076 (to T.W.K.),
National Heart, Lung, and Blood Institute Grant RO1HL66554 (to R.M.T.), the
Flight Attendant Medical Research Institute (S.B.), a Maryland Cigarette Res-
titution Fund research grant (to S.B.), the National Foundation for Cancer
Research (M.B.S.), Reata Pharmaceuticals (M.B.S.), National Institute on Environ-
mental Health Sciences Grants P50ES015903, and ES03819. T.E.S. and D.J.B. are
ES07141, and M.S.Y. is supported by a PhRMA Foundation predoctoral fellow-
ship. H.C.C. is supported by the Bernard A. and Rebecca S. Bernard Foundation.
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