?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 1 January 2012
Angiotensin receptor blockade attenuates
cigarette smoke–induced lung injury and
rescues lung architecture in mice
Megan Podowski,1 Carla Calvi,1 Shana Metzger,1 Kaori Misono,1 Hataya Poonyagariyagorn,1
Armando Lopez-Mercado,1 Therese Ku,1 Thomas Lauer,2 Sharon McGrath-Morrow,2
Alan Berger,3 Christopher Cheadle,3 Rubin Tuder,4 Harry C. Dietz,5
Wayne Mitzner,6 Robert Wise,1 and Enid Neptune1
1Division of Pulmonary and Critical Care Medicine, Department of Medicine, 2Department of Pediatric Pulmonary, 3Johns Hopkins Bayview Lowe Genomics Core,
and 4Division of Cardiopulmonary Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 5Institute of Genetic Medicine,
Howard Hughes Medical Institutes, Johns Hopkins School of Medicine, Baltimore, Maryland, USA. 6Department of Environmental Health Sciences,
Bloomberg School of Public Health, Johns Hopkins School of Public Health, Baltimore, Maryland, USA.
Smoking-related lung diseases, especially chronic obstructive pulmo-
nary disease (COPD) and emphysema, are the third leading cause of
death in the United States. Treatment options are limited to either
symptom relief and/or the elimination of environmental cofactors
such as cigarette smoking. Importantly, despite growing data on the
cellular, molecular, and, recently, genetic features of the disorder,
no novel treatments that can alter the natural history of the disease
are currently available (1). In the studies described here, we extend a
therapeutic approach that has demonstrated efficacy in genetically
defined murine models of airspace enlargement to a murine model of
cigarette smoke–induced (CS-induced) lung injury. Common to these
models are the dual findings of perturbation of the cytokine TGF-β
and airspace enlargement. Therapeutic targeting of TGF-β signaling
in murine models of Marfan syndrome that display progressive air-
space enlargement improves airspace caliber (2, 3). Importantly, we
reported a reversal in airspace enlargement in adult fibrillin-1–defi-
cient mice that were treated over several months with a neutralizing
antibody to TGF-β (2). These findings suggested that antagonism of
TGF-β in lung parenchymal disorders marked by enhanced TGF-β
signaling might provide a reparative milieu for airspace maintenance.
We reasoned that if TGF-β targeting proves effective for murine mod-
els of CS-induced airspace enlargement, we would have proof-of-prin-
ciple evidence that novel translational approaches to COPD can be
garnered from genetically defined animal models with consonant
pathologic, physiologic, and/or biologic features.
The pleiotropic cytokine, TGF-β, has distinct effects on lung
maturation, homeostasis, and repair mechanisms (4, 5). Genetic
association studies of patients with emphysema and histologic
surveys of lungs from patients with COPD of varying severity have
both implicated disturbances in TGF-β signaling as important
components of disease pathogenesis (6). Whereas increased TGF-β
signaling may explain the increased extracellular matrix observed
in the distal airways of patients with severe COPD, reduced signal-
ing with suboptimal matrix deposition might compromise repair
in the airspace compartment, leading to histologic emphysema.
Experimental data support both mechanisms. We recently showed
that fibrillin-1–deficient mice have alveolar septation defects that
are secondary to excessive TGF-β signaling in the airspace compart-
ment (3). We further showed that antagonism of TGF-β signaling
with angiotensin receptor blockade in adult fibrillin-1–deficient
mice with established airspace enlargement improves the airspace
phenotype (2). These data suggest that manipulation of TGF-β sig-
naling might either promote airspace regeneration and/or reduce
airspace destruction. Despite the fact that TGF-β is known to be
dysregulated in COPD/emphysema, TGF-β manipulation has not
been explored in models of CS-induced parenchymal lung disease.
Authorship?note: Megan Podowski, Carla Calvi, Shana Metzger, and Kaori Misono
contributed equally to this work.
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J Clin Invest. 2012;122(1):229–240. doi:10.1172/JCI46215.
230? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 1 January 2012
The role of the renin-angiotensin-aldosterone (RAA) cascade
in the lung is not well described. Apart from known effects on
the microvasculature, reflecting the potent vasoconstrictive
effects of angiotensin II, enhanced RAA signaling also induces
fibrosis in several tissue beds, including the kidney and the
myocardium (7, 8). These latter effects reflect the ability of
angiotensin to promote TGF-β expression and signaling. In
established rodent models of lung injury and fibrosis, angio-
tensin seems to initiate a series of critical TGF-β–dependent
perturbations in the airspace (namely, epithelial cell apoptosis
and epithelial mesenchymal transformation) that cause acute
lung injury and frequently culminate in the fibrotic program.
Importantly, angiotensin receptor blockade attenuates tissue
fibrosis in such model systems (9, 10). Although structural
alveolar apoptosis and airway fibrosis are common features of
COPD pathogenesis, angiotensin receptor blockade has not as
yet been explored in models of COPD/emphysema.
Here we examine the therapeutic utility of TGF-β modula-
tion using two pharmacologic strategies in a murine model of
CS-induced emphysema. We show increased TGF-β signaling
in the lungs of mice exposed to CS and the lung parenchyma
of patients with moderate COPD. Systemic TGF-β antagonism
using either a pan-specific–neutralizing antibody or losartan,
an angiotensin receptor blocker, improves airway and airspace
architecture and lung function in chronic CS-exposed mice,
commensurate with normalized injury measures. These stud-
ies provide compelling preclinical data supporting the utility of
TGF-β targeting for CS-induced lung injury.
Chronic CS induces TGF-β expression in murine lungs and human COPD lungs. (A) TGF-β induction profile by ELISA analysis in lung lysates
from AKR/J mice exposed to 2 weeks of CS. *P < 0.01, CS versus RA or CS plus losartan (Los) versus CS. n = 3–5 mice per treatment group.
(B) Representative histologic sections of lungs from mice exposed to RA or chronic CS subjected to immunohistochemical staining for psmad2.
The inset shows localized staining in alveolar epithelial cells of CS-exposed mice. Arrowheads denote the site of enhanced staining in airspace
(AS) walls of patients with COPD. Original magnification, ×20. n = 4–8 mice per treatment group. (C) Quantitative immunohistochemistry of
psmad2 staining in RA- and CS-exposed mice depicted in B. (D) Representative immunohistochemical staining for total TGF-β1 in lung sec-
tions from a patient with COPD and a control smoker. Original magnification, ×40. Scale bar: 100 μm. n = 10 each of control and COPD tissue
sections. LAP-TGF-β1, latency-associated peptide TGF-β complex. (E) Active TGF-β levels in lung lysates from control nonsmokers (Ctrl –Tob)
(n = 8), control smokers (Ctrl +Tob) (n = 6), and smokers with moderate COPD (COPD +Tob) (n = 11). (F) Representative immunohistochemical
staining for psmad2 in lung sections from a patient with moderate COPD and a control smoker (airspace [2 right panels], airway [left panel]).
(G) Quantitative immunohistochemical staining of psmad2 in airspace compartment and airway compartment in lung sections from patients with
moderate COPD and smoking controls normalized to tissue area. n = 6–11 in each group. AW, airway.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 1 January 2012
Increased TGF-β activity in lungs of mice and lung epithelial cells exposed
to CS and in lungs of patients with COPD. We first evaluated whether
CS exposure resulted in elevated levels of active TGF-β in the lungs
of 2 strains of mice known to be sensitive to CS and whether treat-
ment with the angiotensin receptor blocker losartan normalized
this induction. Two weeks of CS exposure significantly induced
active TGF-β as shown by ELISA analysis in both AKR/J mice (2.5
fold) and C57BL/6 mice (1.4 fold) (Figure 1A and Supplemental
Figure 1A; supplemental material available online with this article;
doi:10.1172/JCI46215DS1). Concurrent losartan treatment nor-
malized TGF-β in both strains. To extend these findings to a chron-
ic CS-induced emphysema model, we evaluated phosphorylated
Smad2 (psmad2) staining, an index of active TGF-β signaling, in
lung sections from mice that develop emphysema after 4 months
of CS exposure, AKR/J mice, and mice that develop emphysema
after 6 months of CS exposure, C57BL/6 mice. Psmad2 staining
was increased in the lungs of both strains of CS-exposed mice (Fig-
ure 1, B and C, and Supplemental Figure 1, B and C), primarily in
alveolar epithelial cells (See inset, Figure 1B). Modest elevations
of connective tissue growth factor (CTGF), a downstream marker
of TGF-β signaling, and TGF-β1 were observed in the lung lysates
from AKR/J mice exposed to 4 months of CS (Supplemental Figure
2). Treatment of murine lung epithelial cells, MLE12 cells, with CS
extract (CSE) also induced enhanced TGF-β activation, evident in
psmad2 expression by immunoblotting (Supplemental Figure 3).
Finally, to extend this observation to clinical COPD, we examined
lung samples from at-risk controls (smokers with normal lung
function) and patients with moderate COPD. ELISA analysis of
active TGF-β1 in lung lysates showed a modest smoking-induced
increase in the whole lung levels that was unaffected by COPD
status (Figure 1E). However, we consistently observed increased
TGF-β1 and psmad2 in the airspaces of patients with moderate
COPD, when compared with those of smoking controls (Figure 1,
D, F, and G). We chose patients with moderate COPD rather than
severe COPD to avoid the end-stage effects often seen with severe
COPD that is punctuated by extensive airspace destruction and
overall reduced protein expression. The TGF-β1 in the lungs of
these patients with COPD was localized to the alveolar septal walls
(similar to that in the murine models) and to inflammatory cells.
These data implicate elevated TGF-β signaling as a component of
CS-induced lung injury.
TGF-β antagonism improves airspace enlargement in chronic CS-exposed
mice. The losartan effect on TGF-β signaling after short-term CS
exposure suggested that angiotensin receptor blockade might have
salutary effects on long-term sequelae of CS exposure. We elected
to use the AKR/J strain in subsequent experiments for 2 reasons:
(a) to incorporate shorter-term chronic exposures that still gen-
erate a measurable airspace lesion and (b) to use an inbred strain
that has a CS-induced inflammatory profile more consistent with
that of a typical patient with COPD than that of the conventional
C57BL/6 model (11). Unfortunately, most investigators still use the
C57BL/6 model that has the potential shortcomings of showing
mild lesions with no evidence of airway pathology when exposed to
CS. To establish the earliest time point at which we could observe
an increase in airspace dimension, the signature feature of emphy-
sema, we exposed AKR/J mice to CS for 1, 2, and 4 months and
performed morphometric analysis. Although no increase in air-
space dimension was observed after of 1 month of exposure, sig-
nificant emphysema developed after 2 months (Figure 2A). (We
also find age-related increases in airspace dimension in room
air–exposed [RA-exposed] mice, a finding we recently dissected in
another inbred strain but notably occurs earlier in the AKR/J mice
[ref. 12].) We treated mice with losartan at 2 doses, 0.6 g/l losartan
(low dose) or 1.2 g/l losartan (high dose) in drinking water, concur-
rent with the CS exposure and found a marked reduction in the
airspace dimension after 2 months (Figure 2, B and C). RA-exposed
mice treated with the 2 doses of losartan showed no change in
airspace caliber or histology compared with those of untreated
controls (Figure 2B and Supplemental Figure 4). Assessment of
airway attachments, a measure of airspace destruction, showed a
significant reduction with CS but recovery with losartan treatment
(Figure 2D). By contrast, CS-induced weight loss was not improved
with either losartan or TGF-β–neutralizing antibody treatment
(Supplemental Figure 5). Losartan treatment of RA-exposed mice
did not alter body weight.
To test the hypothesis that these effects were mediated by inhi-
bition of TGF-β, we treated CS-exposed mice with a neutralizing
antibody to TGF-β (2, 3). Similar to losartan, TGF-β antagonism
with neutralizing antibody given concurrently with CS improved
airspace dimension compared with that of CS-exposed mice treat-
ed with isotype-matched control antibody (Figure 2B). RA-exposed
mice treated with the neutralizing antibody showed no change in
airspace caliber or histology compared with those of untreated
controls (data not shown). Phosphorylated smad2 was increased
in the alveolar and airway epithelium in CS-exposed mice and nor-
malized with losartan treatment (Figure 2, E and F). Thus, two
different strategies targeting TGF-β signaling result in improved
Losartan treatment results in improved lung mechanics and airway his-
tology in chronic CS-exposed mice. The critical disturbance that drives
clinical disease in COPD is the attendant alteration in lung func-
tion that follows from altered lung histology. Compared with those
of RA-exposed mice, CS-exposed mice had increased lung size and
reduced lung elastance, typical physiologic disturbances in emphy-
sema (Figure 3). Losartan normalized lung size and lung elastance,
suggesting that the protective effects apparent by lung histology
translated into improved lung function. Of note, losartan treat-
ment of RA-exposed mice did not significantly alter lung mechan-
ics, although there was a trend toward increased elastance.
Mice exposed to CS developed mucosal thickening that approx-
imated the epithelial hyperplasia observed in patients with
COPD/emphysema (Figure 4A and ref. 13). We measured epithe-
lial thickness in airways of similar size in mice exposed to RA,
CS, CS plus losartan, and CS plus TGF-β–neutralizing antibody.
CS produced a greater than 2-fold increase in airway mucosal
thickness (Figure 4A). Airway epithelial thickening normalized
with losartan treatment and TGF-β–neutralizing antibody treat-
ment. No increase in PAS staining (goblet cells) was observed in
the CS-exposed airways (data not shown). We performed Ki67
staining of the airway compartment to determine whether the
airway thickening represented a proliferative process possibly
triggered by CS exposure. We observed an increase in airway
epithelial proliferation with CS exposure, with a trend toward
reduction with losartan treatment (Figure 4B). Since TGF-β can
induce small airway remodeling, we examined collagen deposi-
tion in CS-exposed lungs. While only a minimal increase in col-
lagen deposition was seen in mice exposed to 2 months of CS,
a marked increase in peribronchiolar collagen deposition was
observed in mice exposed to 3 months of CS (Figure 4C). Losar-
232? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 1 January 2012
tan normalized collagen deposition in such mice. The density
and abundance of αSMA-producing smooth muscle cells sur-
rounding the small airways was not changed with CS or losartan
treatment (data not shown). We thus propose that this airway
lesion is a direct toxic effect of CS and involves TGF-β dysregula-
tion. In summary, airspace enlargement, airway epithelial thick-
ening, peribronchiolar fibrosis, and altered lung mechanics were
all ameliorated by losartan treatment and TGF-β antagonism.
TGF-β antagonism improves CS-induced oxidative stress, inflamma-
tion, and cell death. Oxidative stress and inflammation mediate
CS-induced lung injury in patients with COPD and murine
models of acquired emphysema (14, 15). In AKR/J mice exposed
to 2 weeks or 2 months of CS, nitrotyrosine and 8-deoxyguanine
immunostaining were increased (Figure 5, A and B, and data not
shown), as were alveolar macrophage and lymphocyte numbers
(Figure 5, C and D). Of note, we saw no increase in neutrophils
in the CS-exposed lungs (data not shown). Losartan treatment
normalized oxidative stress and reduced inflammatory cell
infiltration into the CS-exposed lungs (Figure 5, A–D). TGF-β
is known to not only inhibit cellular proliferation, a property
observed in various epithelial model systems, but also induce
cell death, notably in the alveolar lung cells, as seen in fibril-
lin-1–deficient mice (3). We did see reduced airspace epithelial
cell proliferation with CS exposure that did not normalize with
Losartan and TGF-β–neutralizing antibody inhibit chronic CS-induced TGF-β signaling in the lung and attenuate destructive airspace enlargement.
(A) Morphometric analysis of airspace dimension assessed by mean linear intercept (MLI) in mice subjected to 1 month, 2 months, and 4 months
of CS exposure. n = 10–25 mice per treatment group. *P < 0.01. (B) Morphometric analysis of airspace dimension in mice subjected to 2 months
of RA with drinking water or 2 months of CS exposure with drinking water, concurrent low-dose losartan (LD, 0.6 g/l), high-dose losartan (HD, 1.2
g/l), control antibody, or TGF-β–neutralizing antibody (TGFNAb) (10 mg/kg/wk). *P < 0.01, RA versus CS or CS versus CS plus other treatments.
n = 6–8 mice per treatment group. (C) Representative H&E photomicrographs of lungs from mice subjected to 2 months of CS exposure with or
without losartan treatment compared with RA controls. Original magnification, ×20. Scale bar: 200 μm. (D) Airway alveolar attachment count in
mice subjected to the designated treatments. n = 6–8 mice per treatment group. BM, basement membrane. (E) Representative photomicrographs
of lungs subjected to CS compared with RA controls or CS plus losartan stained for psmad2 (brown), a marker of TGF-β signaling (airspace com-
partment [top panel], airway compartment [bottom panel]). Original magnification, ×40. Scale bar: 50 μm. (F) Quantitative immunohistochemistry
of psmad2 staining of lungs from aforementioned treatment groups. n = 6–8 mice per treatment or condition. CS + Los, CS plus losartan.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 1 January 2012
losartan treatment (data not shown). By contrast, the enhanced
TUNEL and active caspase-3 labeling in the airspace, indicating
alveolar epithelial apoptosis, with smoke exposure was attenu-
ated by losartan treatment (Figure 5, E and F).
Effects of losartan on metalloprotease activation and elastin integrity in
the CS airspace. To further assess mechanisms by which elevated
TGF-β might directly induce airspace enlargement, we evaluated
metalloprotease activation and matrix turnover. Zymography
showed increased MMP9, but not MMP2, activation with CS expo-
sure compared with that after exposure to RA. MMP9 activation
was normalized by losartan treatment (Figure 6, A and B). Interest-
ingly, we also observed a modest induction of MMP12 expression
in the lungs of CS-exposed mice that was normalized by losartan
treatment (Figure 6C). We examined elastin fragmentation in the
airspaces of CS-exposed mice and found discontinuous elastin
staining with areas of clumping, which were improved by losar-
tan treatment (Figure 6D). These data suggest that anti–TGF-β
therapy may confer a protective milieu for the extracellular matrix
in the CS-exposed lung.
Taken together, both metalloprotease activation and apoptotic
cell death are the likely underlying mechanisms for the CS-induced
airspace enlargement and are both ameliorated by anti–TGF-β
CS alters angiotensin receptor localization and expression in the murine lung.
Because losartan is a specific angiotensin receptor type 1 (AT1) antag-
onist, we considered whether CS exposure dysregulated AT1 expres-
sion in a manner that enhanced the therapeutic utility of angioten-
sin receptor blockade. Using real-time PCR, we found no differences
in AT1 receptor expression conferred by CS exposure (Figure 7A).
Angiotensin receptors are known to be expressed on lung epithelial
cells, with AT1 localized primarily to the lung parenchyma (16, 17).
Since receptor localization is a critical factor in defining the mecha-
nism of losartan’s effects, we performed immunohistochemistry for
the AT1 receptor on murine lungs subjected to RA, CS, and CS plus
losartan and found that it localized to the alveolar wall and airway
subepithelial mesenchymal layer (Figure 7B). CS increased AT1 stain-
ing in the airspace walls; this was normalized with losartan treatment
(Figure 7, B and inset of B). We propose that the therapeutic losartan
effects that we see in CS-exposed mice may partially reflect increased
expression of angiotensin receptor 1 in the lung parenchyma that is
induced by CS but normalized by losartan.
Transcriptomic signature of therapeutic effect with losartan in CS lung.
The current dearth of rational therapies for COPD/emphysema
prompted us to attempt identification of nonintuitive pathways
that could be exploited for therapeutic targeting. To do this,
we performed an expression profile analysis of lungs from mice
exposed to RA, 2 months CS, or 2 months CS plus losartan. A panel
of genes dysregulated with CS and either further dysregulated or
normalized when treated with losartan was generated (Supple-
mental Figure 6A). We surmised that genes induced or repressed
with CS and then partially or fully normalized with losartan might
represent pathways that contribute to the CS-induced injury phe-
notype. By contrast, we postulated that genes primarily dysregu-
lated with CS and then further dysregulated with losartan likely
reflect reparative pathways triggered with CS exposure and further
reinforced by angiotensin receptor blockade. We found that stress
response and MAPK pathway genes were downregulated with CS
but induced with losartan treatment. Conversely, oxidoreductase,
B cell receptor signaling, chemokine signaling, and cytokine recep-
tor interaction pathways were induced with CS but repressed with
losartan treatment. These findings suggest that whereas survival
pathways may be blunted with CS exposure but restored with
losartan treatment, oxidative stress signaling and immune cell
activation pathways are induced with CS and ameliorated with
losartan treatment. Both profiles are consistent with our dem-
onstration that losartan reduces CS-induced oxidative stress and
inflammation (Figure 4).
To further examine cell survival mechanisms that might be
altered by CS but restored by losartan, we explored TGF–β-induced
pathways that converge onto canonical survival kinase cascades
(p21, p38, JNK, and PI3K/Akt) (18–21). We focused on p21 (pro-
apoptotic/antiapoptotic), p38 (proapoptotic), JNK (proapoptotic),
and akt (antiapoptotic), since these can be modulated by TGF-β.
Since signaling measurements using total lung lysates are reflec-
tive of the composite of the multiple compartments present in
the lung parenchyma rather than the site of relevant activity, we
elected to use both immunoblotting and in situ surveys to assess
prosurvival signaling with CS exposure and losartan treatment.
We saw no evidence of p21 induction or activation, respectively,
in CS-exposed lungs (data not shown). We observed attenuated
Akt, JNK, and p38 activation by immunoblotting in CS-exposed
lungs (Supplemental Figure 6B). However, only Akt activation
was normalized by losartan treatment (Supplemental Figure 6C).
These data suggest that a candidate mechanism by which losartan
improves airspace dimension is by enhancing Akt-mediated pro-
survival signaling and reducing alveolar apoptosis. We examined
the distribution of akt staining in the lung and found localization
in the airspace epithelial cells (Supplemental Figure 6, D and E).
The reduction in staining in the airspace compartment with CS
suggested that the immunoblotting pattern reflected events at the
site of known CS-induced lung pathology.
Effect of losartan treatment on lung mechanics of CS-exposed mice.
Total lung capacity of lungs subjected to designated treatments (top).
Static lung elastance of mice subjected to designated treatments (bot-
tom). *P < 0.05 for CS compared with RA; **P < 0.05 for CS and losar-
tan compared with CS. n = 6–8 mice per treatment or condition. Data
are represented as mean ± SEM.
234? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 1 January 2012
The role of TGF-β dysregulation in CS-induced COPD/emphy-
sema is a controversial issue, given abundant but conflicting data
showing evidence of both enhanced and reduced activity in the
COPD lung. We show here evidence of increased TGF-β activity
in the airspaces of chronic CS-exposed mice and patients with
mild COPD. We further establish that pharmacologic inhibition
of TGF-β signaling protects the murine lung from altered lung
histology, impaired lung function, and a panel of injury measures
that accompany CS-induced lung disease. Whereas emphysema
was originally thought to solely require elastin destruction, the
current pathogenetic schema incorporates additional mecha-
nisms, such as cell death and oxidative stress injury (22, 23).
Importantly, the pleiotropic effects of TGF-β signaling impact
on all of these contributing mechanisms. This study provides
compelling preclinical evidence for the utility of TGF-β targeting
for common and complex CS-promoted lung pathologies, such as
COPD/emphysema and respiratory bronchiolitis.
TGF-β signaling incorporates a large family of ligands, cell-
surface receptors, and coreceptors that engage a complex but
canonical cascade of intracellular mediators to modulate tis-
sue morphogenesis and repair. TGF-β has multiple functions
in the airspace, a compartment composed of multiple cell types
of endodermal, mesenchymal, vascular, and hematopoietic lin-
eage. The response to TGF-β in each of these cell types is dis-
tinct and context dependent (reviewed in ref. 24). What is clear
Airway wall thickening and epithelial hyperplasia in chronic CS-exposed mice. (A) Representative H&E images of small airways from mice treated
with 2 months of RA, CS, CS plus losartan, or CS plus TGF-β–neutralizing antibody (TGFNAb). Original magnification, ×20. Scale bar: 50 μm.
Measurement of airway wall thickness of small airways of similar caliber in mice subjected to designated treatments. Data are expressed as mean ±
SEM. **P < 0.01. n = 6–8 mice per treatment. (B) Representative lung sections of airways from mice in designated treatment groups stained for pro-
liferation marker Ki67. n = 4–6 mice per group. Original magnification, ×20. Scale bar: 100 mm. Quantitative immunohistochemistry of Ki67 staining
of airway epithelial cells. (C) Representative images of trichrome staining of airways from mice in designated treatment groups. Original magnifica-
tion, ×20. Scale bar: 100 μm. Quantitation of trichrome staining in designated groups normalized to airway perimeter. n = 7–9 mice per group.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 1 January 2012
is that the homeostatic level of TGF-β is well maintained, and
interventions directed toward correcting excess in either direc-
tion are reasonable strategies. Although TGF-β can induce
fibroblast cell differentiation into highly synthetic myofibro-
blasts and arguably transdifferentiation of epithelial cells into
fibroblasts, the pathway can have prominent antiproliferative
and proapoptotic effects in the epithelial compartment (14, 25).
Our observation of a prominent proapoptotic effect in the air-
space epithelial compartment of CS-exposed lungs accompany-
ing peribronchial fibrosis is consistent with a TGF-β–mediated
profile. However, TGF-β effects in most tissues are dictated by
both cellular context and signaling intensity, with a physiologic
window defined by the optimal level of ambient ligand abun-
dance and cellular capacity for response. The selective epithe-
lial and peribronchiolar response to TGF-β signaling in our
model suggests that chronic CS induces an elevation of TGF-β
sufficient to compromise epithelial cell survival and promote
submucosal fibrosis in the distal airway but not to induce an
interstitial fibrotic program. Of note, most TGF-β transgenic
overexpression maneuvers in the lung result in exuberant path-
way activation and therefore culminate in parenchymal fibrosis
(26, 27). However, selective TGF-β–overexpressing mice as well
as nonfibrotic rodent injury models associated with elevated
TGF-β levels consistently show early airspace enlargement with
Effect of losartan on CS-induced injury measures. (A) Nitrotyrosine (NiTyr) staining (brown) of lung parenchyma (right) and airways (left) of lungs
exposed to CS or CS plus losartan. Original magnification, ×40. Scale bar: 50 μm. n = 4–6 mice per group. (B) Quantitative immunohistochemistry
of nitrotyrosine-stained lungs. Staining was normalized to tissue area. n = 4–6 mice per group. (C) Quantitative immunohistochemistry of
macrophage abundance in lungs using MAC3 staining. Staining was normalized to tissue area. n = 4–6 mice per group. (D) Quantitative
immunohistochemistry of lymphocyte abundance in lungs using CD45R staining. Staining was normalized to tissue area. n = 4–6 mice per
group. (E) Representative photomicrographs of TUNEL-stained lungs. Arrowheads denote staining in airspace epithelial cells in CS-exposed
lungs. Original magnification, ×20 (top row); ×40 (bottom row). Scale bar: 50 μm. n = 4–6 mice per condition or per treatment. Quantitative
immunohistochemistry of TUNEL staining reflecting the apoptotic index. Data are represented as mean ± SEM. (F) Representative photomicro-
graphs of active caspase-3–stained (C3-stained) lungs. The black arrowhead denotes positive staining in type II alveolar epithelial type II cell.
The white arrowhead denotes negative staining in nearby type II epithelial cell. The black arrow shows lack of staining in type I alveolar epithelial
cell. Original magnification, ×40. Scale bar: 50 μm. n = 4–6 mice per condition or per treatment. Quantitative immunohistochemistry of active
caspase-3 staining normalized to tissue area. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01.
236? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 1 January 2012
variable components of mild fibrosis (28–30). Thus, the com-
partmentalized fibrotic effects of CS-induced TGF-β activity are
fully consistent with other rodent models systems punctuated
by injury-associated airspace enlargement.
What is the evidence for TGF-β dysregulation in human COPD/
emphysema? Compelling genetic data from multiple laboratories
implicate disturbances in TGF-β signaling in COPD pathogenesis.
However, the nature of the disturbance, too high or too low, is a
subject of controversy. In several studies, TGF-b1 polymorphisms
associate not only with the diagnosis of COPD but also with disease
severity (31–34). However, other studies have not validated such asso-
ciations (33, 35). Recently, polymorphisms in a TGF-β binding pro-
tein (LTBP) and a TGF-β coreceptor (betaglycan) were found to asso-
ciate with distinct COPD-related subphenotypes (31, 36). Although
a connection between TGF-β polymorphisms and serum levels was
initially presumed based on a few publications, subsequent studies
in larger and more heterogeneous populations have not consistently
shown this association (37–40). Immunohistochemical studies of
COPD lung specimens show evidence of enhanced TGF-β signaling
predominantly in the airway compartment (41–43). Gene expression
studies from lung specimens of patients with COPD demonstrate
enhanced activation of TGF-β pathways that may well be stage and
compartment dependent (44–46). Interestingly, selective animal
models with defects in TGF-β signaling have also shown develop-
mental or late-onset airspace enlargement (47–49). These seemingly
conflicting findings suggest that a critical level of TGF-β signaling
is required for airspace formation and maintenance and that dis-
orders resulting in either marked excess or profound deficiency in
TGF-β signaling translate into abnormal airspace architecture. Fur-
thermore, the activation of compensatory mechanisms that serve to
enhance TGF-β signaling might be operative in these models (50).
Thus, dysregulated TGF-β signaling provides a unifying explanation
for the divergent manifestations of COPD with cellular prolifera-
tion with fibrosis in terminal airways and apoptotic cell death in the
alveolar compartment. Our data establish for what we believe to be
the first time that enhanced TGF-β activity is not merely a signature
of COPD but contributes to disease pathogenesis.
We demonstrate an intriguing and previously unreported air-
way epithelial phenotype that approximates the epithelial hyper-
plasia that can accompany a variety of airway insults, including CS
Effects of losartan on matrix metalloprotease activity and expression. (A) Zymography of lung extracts from representative mice with designated
exposures and treatments. The top band (black arrowhead) denotes MMP9, and the lower band (gray arrowhead) denotes MMP2. The positive
(+) control data represents recombinant mouse MMP9. The lanes were run on the same gel but are noncontiguous. n = 4–8 mice per treatment.
(B) Densitometry of MMP9 zymography bands. n = 4–8 mice per treatment. (C) Western blot analysis of MMP12 expression in lung lysates
from mice exposed to RA, CS, or CS plus losartan. MMP12 and β-actin bands are shown. n = 4–6 mice per condition. (D) Elastin localization
by Hart’s stain with and without tartrazine counterstaining. Arrows in the top and middle rows show linear deposition of elastin in alveolar walls
of RA-exposed mice, and arrowheads show dense, discontinuous deposition in walls in CS-exposed mice. The latter is improved with losartan
treatment (arrow). Note that pale staining in airspaces reflects residual agarose in lungs. Scale bar: 50 μm. n = 4–6 mice per condition.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 1 January 2012
(reviewed in ref. 51). Airway wall thickening is a complex pathol-
ogy in clinical COPD but seems to be a consequence of excessive
TGF-β activation (42, 52). Whether submucosal matrix deposi-
tion, airway epithelial thickening, or mucus hypersecretion is the
critical pathologic lesion that accounts for clinical obstruction is
debatable (13). Murine models typically display modest airway
wall remodeling in response to chronic CS, an observation that
is thought to be a consequence of the anatomic and cell composi-
tional differences between the rodent and the primate airway (53).
Nonetheless, clinical hyperexpansion with air trapping is a direct
consequence of the airway lesion and is associated with accelerated
lung function decline in patients with emphysema (54). Accord-
ingly, in our model, the increased lung volumes likely follow from
the airway mucosal thickening. A recent small clinical trial of
angiotensin receptor blockade in patients with COPD similarly
showed improvement in lung hyperexpansion with 4 months of
treatment (55). Thus, the data generated in our preclinical model
approximate effects observed in small studies of this agent in a
comparable clinical population.
The current report suggests that enhanced TGF-β is a therapeutic
point of convergence for the inflammation, oxidative stress, cell death,
and, importantly, metalloprotease activation associated with chronic
CS exposure. Metalloprotease activation causing matrix turnover is a
fundamental mechanism of COPD development and maintenance.
Polymorphisms in MMP12 associate with reduced lung function in
patients with COPD and children with asthma (31). Mice deficient in
MMP12 are protected against CS-induced emphysema
(56). However, the role of TGF-β signaling in metal-
loprotease expression and activation is highly con-
textual, with evidence of inductive effects on MMP9
and inhibitory effects on MMP12 (57–59). Further,
reduced TGF-β signaling seems to punctuate some
models of aging-related airspace enlargement, possibly
secondary to both a temporally defined impairment
in maintenance elastogenesis and elevated MMP12
expression (47, 48). The scenario with CS appears
distinct from this process. Since TGF-β can induce
MMP9 expression and MMP9 can activate TGF-β, our
observed pattern of MMP9 activation is consistent
with a TGF-β–mediated process (60–62). Whereas in
the aging-associated airspace enlargement models,
TGF-β is thought to inhibit MMP12 expression in
macrophages, our seemingly paradoxical result may
reflect a direct effect of CS exposure on the proposed
regulatory scheme and/or the enhanced macrophage
abundance in the lungs of CS-exposed mice (47).
Even though airspace maintenance in the setting of CS expo-
sure must converge upon known cell injury and cell death pro-
cesses, the role of CS on prosurvival signaling in the airspace
has not been well dissected. Our studies provide some insight
into these cascades. Using a combination of whole tissue and in
situ analysis, we implicate reduced Akt signaling as potential-
ly involved in the alveolar septal cell survival disturbance that
culminates in enhanced cell death observed in the chronic CS
model. Others have shown that Akt signaling is a critical media-
tor of airspace homeostasis in the setting of neonatal and adult
hyperoxic injury (63, 64). Furthermore, several in vitro studies
demonstrate that TGF-β directly inhibits Akt-mediated lung epi-
thelial cell survival (65, 66). We show that a similar mechanism
may be operative with chronic CS-induced lung injury. The fur-
ther dissection of Akt signaling in airspace repair is a fertile area
of research for our laboratory.
We elected to forgo the use of the most common CS-induced
emphysema model, the C57BL/6 mouse, in preference of
the AKR/J strain. This choice was based on compelling data,
suggesting that the C57BL/6 model may be inferior to other
strains, including AKR/J mice, with respect to the airspace
lesion, inflammatory milieu, and effects on lung mechanics
conferred by CS exposure (11, 67). Of particular concern with
the C57BL/6 mouse is that the need for more than 6 months of
CS exposures often results in the complicating phenotype of
aging-associated airspace enlargement.
Angiotensin receptor expression in CS-exposed lungs.
(A) Real-time PCR quantitation of AT1 (Agtr1a) expres-
sion in CS- and CS plus losartan–treated mice com-
pared with that in RA controls. Receptor expression
was normalized to Gapdh. Error bars represent SEM.
n = 4–6 mice per treatment group. (B) Representative
lung sections stained for AT1 (black) in adult mice sub-
jected to 2 months of RA, CS, or CS plus losartan. The
arrowhead in the inset denotes enhanced staining for
AT1 in the airspace wall of CS-exposed mice. Scale
bar: 50 μm; 25 μm (inset). n = 4–6 mice per treatment
or condition. Data are represented as mean ± SEM.
238? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 1 January 2012
We report a murine model of CS-induced lung disease that
manifests both airway wall thickening and airspace simplification
after 2 months of smoke exposure. This model displays increased
TGF-β signaling and oxidative stress and inflammation in the
airway and alveolar compartments. Altered cell survival signaling
culminates in increased alveolar cell death. More importantly, we
show that systemic antagonism of TGF-β signaling with angioten-
sin receptor blockade normalizes histology and reduces oxidative
stress, cell death, and inflammation. Pulmonary function stud-
ies show improved lung mechanics with losartan treatment. An
exploratory transcriptional survey implicates the involvement of
immunomodulatory and stress response pathways in the thera-
peutic effects of losartan. Our findings provide a preclinical plat-
form for the development of other TGF-β–targeted interventions
in translational approaches to COPD/emphysema.
Mice. Adult AKR/J mice were obtained from The Jackson Laboratory.
These mice were housed in a facility accredited by the American Associa-
tion of Laboratory Animal Care, and the animal studies were reviewed and
approved by the institutional animal care and use committee of Johns
Hopkins School of Medicine.
CS exposure. Six- to eight-week-old AKR/J male mice were divided into 3
groups. The control group was kept in a filtered air environment, and the
experimental groups were subjected to CS or CS plus losartan in drinking
water. CS exposure was carried out (2 hours per day, 5 days per week) by
burning 2R4F reference cigarettes (University of Kentucky, Louisville, Ken-
tucky, USA) using a smoking machine (Model TE-10; Teague Enterprises)
for 6 to 7 weeks. The average concentration of total suspended particulates
and carbon monoxide was 90 mg/m and 350 ppm, respectively, which was
monitored on a routine basis.
Human studies. All human lung tissue from persons with COPD and at-
risk controls were obtained, as anonymized samples, from the Lung Tissue
Research Consortium (LTRC; http://www.nhlbi.nih.gov/resources/ltrc.
htm), sponsored by the National, Heart Lung and Blood Institute. Based
on spirometry and smoking history, the patients were designated as at-risk
(>10 pack year history of smoking; normal spirometry) or as having moder-
ate or severe COPD using Global Initiative for Chronic Obstructive Lung
Disease (GOLD) criteria (moderate, GOLD, 2; forced expiratory volume at
1 second [FEV1], 50%–80% predicted; severe, GOLD, 3 and 4; FEV1, <50%
predicted) (68). All smokers were former smokers.
Cell treatment. MLE12 cells (ATCC) were treated with CSE for 72 hours
after serum starvation overnight. CSE was generated per standard protocol
by the D’Amico laboratory, Johns Hopkins School of Medicine (69). Cell
lysates were harvested and subjected to immunoblotting for psmad2 (Cell
Treatment regimen. The AT1 selective antagonist losartan (Merck Co.) was
diluted into drinking water at concentrations of 3 mg/kg (low dose) and
30 mg/kg (high dose). Panselective TGF-β–neutralizing antibody (R&D
Systems) was administered by intraperitoneal injection according to pub-
lished protocol (70). Isotype-matched control antibody (R&D Systems)
was administered to control mice as described above.
Morphology and histology. Three to five mice of each genotype were stud-
ied at the noted ages. For histologic and morphometric analyses, mouse
lungs were inflated at a pressure of 25 cm H2O and fixed with 4% PFA
in low molecular weight agarose. The lungs were equilibrated in cold
4% PFA overnight, sectioned, and then embedded in paraffin wax. Sec-
tions were cut at 5 μm and either stained with H&E or processed for
immunohistochemistry. For the human lung samples, 2–3 slides from
each patient or control were used for analysis.
Morphometry and histochemistry. Mean linear intercept measurements were
performed on H&E-stained sections taken at intervals throughout both
lungs. Slides were coded, captured by an observer, and masked for identity
for the groups. Ten to fifteen images per slide were acquired at ×20 mag-
nification and transferred to a computer screen. Mean chord lengths and
mean linear intercepts were assessed by automated morphometry with a
macro-operation performed by Metamorph Imaging Software (Universal
Imaging, Molecular Devices). Mean airway thickness was measured directly
using microscope-captured images at ×40 magnification. Hart’s staining
was performed per published protocol using either van Gieson or tartra-
zine counterstaining (71).
Immunoblotting. Whole lung lysates were extracted in M-Per buffer
from Pierce. Protein concentrations were determined using the Bio-
Rad Protein Assay. Aliquots of 30–50 μg protein were boiled and then
loaded onto Tris-HCL gels and transferred electrophoretically to nitro-
cellulose membranes. Membranes were incubated with the primary
antibody for 1 hour at room temperature. Detection was performed by
the Pierce West Dura ECL Detection System. Primary antibodies and
dilutions were as follows: β-actin (rabbit polyclonal, 1:1,000; Abcam),
p38 (rabbit polyclonal, 1:1,000; Cell Signaling Technology), pp38 (goat
polyclonal, 1:200; Cell Signaling Technology), ERK1 (rabbit polyclonal,
1:1,000; Cell Signaling Technology), pERK1 (rabbit polyclonal, 1:1,000;
Cell Signaling Technology), JNK (rabbit polyclonal, 1:1,000; Cell Sig-
naling Technology), and pJNK (rabbit polyclonal, 1:1,000; Cell Signal-
Immunohistochemistry. For details regarding protocol, please see the Sup-
plemental Methods. Briefly, after incubation with the primary antibody
overnight at 4°C, slides were washed with PBST, incubated with an appro-
priate biotinylated secondary antibody (Jackson ImmunoResearch Inc.),
and developed by using ABC and DAB detection reagents (Vector Labora-
tories). Antibodies were used at the following concentrations: Ki67 (1:50;
Santa Cruz Biotechnology Inc.), nitrotyrosine (Abcam), Mac3 (BD Biosci-
ences), CD45R (Santa Cruz Biotechnology Inc.), psmad2 (Cell Signaling
Technology), TUNEL (1:25; Abcam), JNK/pJNK (Cell Signaling Technol-
ogy), Akt/pAkt (Cell Signaling Technology), LAP-TGF-β1 (R&D Systems),
CTGF (Abcam), Angiotensin type 1 receptor (Santa Cruz Biotechnology
Inc.), and active caspase-3 (Abcam).
Measurement of mouse lung mechanics. Mice were anesthetized with a
ketamine (90 mg/kg)/xylazine (18 mg/kg) mixture. Once sedated, a trache-
ostomy was performed, and a cannula (18G) was inserted and connected to
a constant flow ventilator as previously described (72).
Quasistatic PV curves were performed as previously reported (73). Details
regarding protocol are in the Supplemental Methods.
Statistics. One-way ANOVA with Tukey’s post-hoc test or Kruskal-Wal-
lis nonparametric analysis with a Dunnett’s post-hoc test were used to
determine differences among groups. When 2 groups were compared, an
unpaired, 2-tailed Student’s t-test or a Wilcoxon rank-sum test was used.
Values for all measurements were expressed as mean ± SEM, and P values
for significance were less than 0.05. The number of samples or animals in
each group is indicated in the figure legends or text.
Study approval. For the LTRC specimens, all patients provided informed
consent to the LTRC. We confirmed IRB-exempt status for these studies
with the Johns Hopkins Office of Human Subjects Research (study no.
We thank Robert Senior, Dean Sheppard, Ari Zaiman, and Land-
on King for helpful comments. We thank the Cytokine Core
Laboratory at University of Maryland for the ELISA analyses.
We thank the Biswal laboratory for supervising the CS expo-
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 1 January 2012
sure at the Core Facility in the Johns Hopkins School of Public
Health. We thank the D’Amico laboratory for the provision of
CSE. We thank Tracy Adair-Kirk for help with the Hart’s stain
assay. We thank Norman Barker for help with figure prepara-
tion. This work was supported by NIH grants R01HL085312
and R03HL095406-01 (to E.R. Neptune) and the NIH National
Heart, Lung and Blood Institute Specialized Centers of Clini-
cally Oriented Research Grant P50HL084945 (to E.R. Neptune,
R. Wise, W. Mitzner, and S. Metzger). We would like to acknowl-
edge the generous support of the Grace Anne Dorney fund for
Received for publication April 18, 2011, and accepted in revised
form November 9, 2011.
Address correspondence to: Enid R. Neptune, Room 547, 1830
East Monument Street, Johns Hopkins University School of Medi-
cine, Baltimore, Maryland 21205, USA. Phone: 410.955.4176; Fax:
410.955.0036; E-mail: firstname.lastname@example.org.
Rubin Tuder’s present address is: Program in Translational Lung
Research, University of Colorado School of Medicine, Aurora,
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