Pulmonary Epithelial Neuropilin-1 Deletion Enhances
Development of Cigarette Smoke–induced Emphysema
Anne Le1, Rachel Zielinski1, Chaoxia He1, Michael T. Crow1, Shyam Biswal1,2, Rubin M. Tuder1,3,
and Patrice M. Becker1
1Divisions of Pulmonary and Critical Care Medicine and3Cardiopulmonary Pathology, Johns Hopkins University School of Medicine, Baltimore,
Maryland; and2Division of Toxicology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland
Rationale: Cigarette smoke (CS) exposure is an important risk factor
develop disease, suggesting that other factors influence disease
integral component of receptor complexes mediating alveolar
septation and vascular development, was involved in maintenance
of normal alveolar structure, and/or altered susceptibility to the
effects of CS.
specific deletion of epithelial Nrp1. We determined whether condi-
and the balance between cell death and proliferation in condition-
ally Nrp1–deficient adult mice and littermate controls. Finally, we
evaluated the effects of Nrp1 silencing on cell death after acute
exposure of A549 cells to cigarette smoke extract or short chain
I and type II alveolar epithelial cells were significantly enhanced
following chronic CS exposure in conditionally Nrp1-deficient adult
mice. Silencing of Nrp1 in A549 cells did not alter cell survival after
vehicle treatment but significantly augmented apoptosis after
exposure to cigarette smoke extract or ceramide.
Conclusions: These data support a role for epithelial Nrp1 in the
maintenance of normal alveolar structure and suggest that dysre-
gulation of Nrp1 expression may promote epithelial cell death in
response to CS exposure, thereby enhancing emphysema develop-
Keywords: chronic obstructive pulmonary disease; genetically modi-
fied mice; apoptosis
Chronic obstructive pulmonary disease (COPD), defined by the
presence of chronic bronchitis and/or emphysema, is a progres-
sive disorder of the airways, characterized by a gradual loss of
lung function. At least 24 million adults nationally are estimated
to carry this diagnosis, which has been the fourth leading cause
of death in the United States since 1991 (1). The single most
prevalent and preventable risk factor for the development of
COPD is cigarette smoke (CS) exposure (2, 3), which accounts
for more than 80 to 90% of the COPD cases in the U.S. (4).
Only approximately 20% of smokers develop clinical evidence
of COPD (5, 6), suggesting that other factors, including genetic
predisposition, may influence disease development.
Neuropilin is a type I membrane protein that is highly
conserved across vertebrate species (7–9). Two members of
the neuropilin family have been identified, neuropilin-1 (Nrp1)
and neuropilin-2 (Nrp2) (7). Both were initially identified as
specific neuronal receptors for a class of secreted signaling
proteins, the class 3 semaphorins (Sema3), which guide axonal
growth cone collapse (7, 10–12). Neuropilins are also widely
expressed in nonneuronal tissue, including epithelium and
endothelium, and Nrp1 gene expression has been described in
tissue homogenate from lung, heart, placenta, skeletal muscle,
kidney, pancreas, liver, and brain (13, 14).
Nrp1 was independently cloned from neurons as a receptor
for Sema3 (7, 11) and from endothelium as a novel vascular
endothelial growth factor (VEGF) receptor (14, 15). In the
nervous system, Nrp1 mediates semaphorin-induced axonal
growth cone collapse (7, 11) and is required for semaphorin-
mediated neuronal (16) and neural progenitor cell apoptosis
(17). In endothelium, Nrp1 enhances VEGF-mediated phos-
phorylation and signaling via VEGFR2 (18–20) and enhances
VEGF-induced endothelial permeability (18), chemotaxis (18,
19), proliferation, and cell survival (19). Emerging data suggests
that although semaphorins can modulate endothelial signaling
by competitively inhibiting VEGF signaling (21), they may have
independent effects on endothelial function (22, 23).
Several groups have described abundant expression of Nrp1
in pulmonary epithelium (24, 25), yet little is known about the
function of Nrp1 in epithelial cells. Nrp1 may modulate the
balance between VEGF and Sema3 signaling to alter cell
proliferation and death in nonepithelial cell types (17, 26). Both
AT A GLANCE COMMENTARY
Scientific Knowledge on the Subject
An imbalance between pathways mediating alveolar cell
proliferation and death may lead to airspace destruction
and the generation of emphysema. Disruption of growth
factor signaling cascades critical for lung development and
postnatal homeostasis may promote the development of
emphysema in response to cigarette smoke exposure.
What This Study Adds to the Field
This study suggests that loss of pulmonary epithelial Nrp1
enhances apoptosis of Type I and II epithelial cells and
airspace enlargement in response to chronic cigarette
(Received in original form September 23, 2008; accepted in final form June 9, 2009)
Supported by a Clinical Innovator Award from the Flight Attendant Medical
Research Institute (052365; P.M.B.), National Institutes of Health (NIH) grant
RO1HL083286 (P.M.B.), NIH RO1HL081205 (S.B.), SCCOR P50HL084945
(R.M.T., S.B.), NIH R01HL066554 (R.M.T.), and the Alpha 1 Foundation (R.M.T.).
Correspondence and requests for reprints should be addressed to Patrice M.
Becker, M.D., Division of Pulmonary and Critical Care Medicine, Johns Hopkins
Asthma and Allergy Center, Room 4B74, 5501 Hopkins Bayview Circle, Baltimore,
MD 21224. E-mail: email@example.com
This article has an online supplement, which is accessible from this issue’s table of
contents at www.atsjournals.org
Am J Respir Crit Care Med
Originally Published in Press as DOI: 10.1164/rccm.200809-1483OC on June 11, 2009
Internet address: www.atsjournals.org
Vol 180. pp 396–406, 2009
Nrp1 ligands, VEGF, and Sema3A, have independently been
shown to contribute to alveolar septation (24, 27–30) and
vascular patterning (23, 27, 31), and human (32, 33), animal
(34–37), and in vitro (38) studies support a possible role for
VEGF in protection from acute lung injury and preservation of
alveolar cell survival (39). Of note, it was shown that expression
of Nrp1 protein was reduced in the lungs from smokers with
spirometric evidence of COPD compared with smokers who
had normal lung function and nonsmoking control subjects (40).
Because Nrp1 is part of essential receptor complexes mediating
both VEGF and Sema3A signals, we hypothesized that epithe-
lial Nrp1 might play a role in the maintenance of normal
To test this hypothesis, we generated transgenic mice in
which tissue-selective conditional deletion of Nrp1 in the
pulmonary epithelium could be achieved, and determined the
effects of lung epithelial Nrp1 deletion on lung morphometry
during either the postnatal period or adulthood. Because this
series of experiments demonstrated that loss of epithelial Nrp1
resulted in a phenotype of mild airspace enlargement, we then
evaluated whether pulmonary epithelial Nrp1 deletion in adult
mice enhanced the injurious effects of chronic CS exposure,
promoting alveolar epithelial cell death and the development of
emphysema in these animals. Finally, in vitro experiments were
performed to confirm a role for Nrp1 in alveolar epithelial cell
survival. Some of the results of these studies have been pre-
viously reported in the form of abstracts (41, 42).
Genetically Modified Mice
Animal studies were approved by the Johns Hopkins Animal Care and
Use Committee. Inducible lung cell-specific deletion of Nrp1 was
of mice for these experiments are provided in the online supplement.
Effects of Conditional Pulmonary Epithelial Nrp1 Deletion
in Adult Mice
Inducible pulmonary epithelial Nrp1 deletion was achieved by admin-
istration of doxycycline-containing chow (0.625 g/kg; Harlan-Teklad,
Madison, WI). To determine the effects of neonatal Nrp1 deletion, chow
was administered to CCSP-rtTA/tetO-Cre/Nrp1flox/floxmice and litter-
mates at Postnatal Day 7 (P7), and continued for 12 weeks before killing
(n 5 5–7/group). To assess the effects of pulmonary epithelial Nrp1
deletion after postnatal alveolarization was complete, adult CCSP-rtTA/
tetO-Cre/Nrp1flox/floxmice and littermate controls received doxycycline-
containing chow beginning at 9 to 10 weeks of age and continuing for 12
weeks before killing (n 5 6/group).
Chronic CS Exposure
CS exposure was performed as previously described (44) and outlined in
the online supplement. Briefly, at 10 weeks of age CCSP-rtTA/tetO-Cre/
Nrp1flox/floxmice (n 5 16) and littermate controls (n 5 17) were divided
into two groups. Half of the animals of each genotype were exposed
simultaneously to CS (5 h/d, 5 d/wk) and doxycycline-containing chow
for 12 weeks. The remaining mice served as controls and were main-
tained on the same chow in a filtered air (FA) environment for the
specified time period.
For histologic analysis, animals were anesthetized, the trachea cannu-
lated, and the right hilum ligated, then the right lung was freeze-
clamped in liquid nitrogen for biochemical studies, and the left lung
was inflated for morphometry and immunohistochemical analysis (see
online supplement). Lung inflation was performed with 0.5% low-
melting agarose at a constant pressure of 25 cm H2O, as previously
described (45). Four-millimeter transversal sections of the left lung were
then fixed in toto in 4% paraformaldehyde overnight and subsequently
embedded in paraffin. Five-micrometer sections were stained with
hematoxylin and eosin. The lung sections in each group were randomly
coded, and representative images (20 per lung) were acquired with
a Nikon E800 microscope (lens magnification 103; 100–200 alveoli per
field) by an investigator who was masked to the identity of the slides.
Mean linear intercept (MLI), mean chord length (MCL), and surface to
volume ratio (S:V) were determined by computer-assisted morphometry
with MetaMorph (Molecular Devices, Downington, PA).
Detection of Cell Proliferation and Apoptosis after
FA and CS Exposure
Cell turnover was evaluated by immunostaining lung sections with an
antibody recognizing proliferating cell nuclear antigen (PCNA; Santa
Cruz Biotechnology, Santa Cruz, CA), and an antibody that recognizes
the cleaved (17/19 kD) fragment of activated caspase 3 (Cell Signaling
Technology, Boston, MA). Lung sections were randomly coded, and
the relative expression of both PCNA and active caspase 3 was
determined by normalizing the number of PCNA- or caspase 3-positive
cells to the total cell number, identified by nuclear staining with 49-69-
diamidino-2-phenylindole (DAPI). At least 10 random fields (603)
were captured from the distal lung parenchyma of each mouse, and
a minimum of 500 nuclei were counted for each lung. Staining was
quantified by an investigator blinded to the identity of the slides
relative to experimental group.
To determine the cell type undergoing apoptotic cell death,
terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling
(TUNEL) was performed. After antigen retrieval, the TUNEL reaction
was performed by incubating the sections with 96 ml equilibration buffer,
2 ml biotinylated nucleotide mix, and 2 ml rTdT enzyme (Promega,
Madison, WI) for 1 hour at 378C followed by incubation with Alexa
Fluor 647 streptavidin conjugate (Invitrogen, Carlsbad, CA) for 1 hour
at room temperature. Type I and II epithelial cells were detected by
coimmunostaining with hamster monoclonal anti-T1a (kindly provided
by Dr. Mary Williams) and rabbit anti-prosurfactant Protein C (Chemicon,
San Diego, CA), respectively. Control sections were incubated with
nonimmune IgG in place of the primary antibody. Slides were randomly
coded, analyzed using a Zeiss LSM 510 META confocal microscope at
1003 magnification, and five random fields from each slide were
captured for analysis. Quantification was performed by an investigator
masked to the identity of the slides relative to experimental group.
Protein Expression and Activity
Measurement of VEGF protein in tissue lysates was performed using
a commercially available sandwich ELISA (R&D, Minneapolis, MN).
Expression of Nrp1, VEGFR2 and phosphorylated VEGFR2 (p-
VEGFR2) was determined using Western blot analysis, using primary
antibodies from Santa Cruz Biotechnology (VEGFR2, p-VEGFR2), or
provided by David Ginty (Nrp1 ). Expression of p-VEGFR2 was
normalized to total VEGFR2 expression, and Nrp1 and VEGFR2
expression were normalized to expression of GAPDH on the same
Following FA or CS exposure, MMP-2 and MMP-9 activity were
assessed using gelatin zymography (n 5 3/group; see online supple-
ment). MMP-12 activation (n 5 4–6/group) was determined indirectly
by Western blot analysis, using primary antibodies (Santa Cruz Bio-
technology) that detect both full-length and cleaved MMP-12.
A separate series of adult CCSP-rtTA/tetO-Cre/Nrp1flox/floxmice and
their littermates were exposed to 12 weeks of CS or FA (n 5 4–5/
group), then lung lavage was obtained by the gentle instillation then
aspiration of 0.3 ml (33) phosphate buffered saline (378C). Total cell
count was obtained using a hemocytometer, a cytospin was performed
to assess differential cell count, and the remaining fluid was centrifuged
and supernatant frozen at 2808C for measurement of VEGF protein
levels with a commercially available sandwich ELISA (R&D Systems,
Inflammatory Mediator Expression in Lung Tissue
Tissues from FA- and CS-exposed CCSP-rtTA/tetO-Cre/Nrp1flox/flox
mice and littermate controls (n 5 6–7/group) were homogenized in
Le, Zielinski, He, et al.: Neuropilin-1 and Emphysema397
Tris buffer containing protease inhibitor cocktail (Sigma Chemical,
St. Louis, MO) and phosphatase inhibitors Na3VO4(100 mM) and NaF
(50 mM), then protein concentration was determined (Bio-Rad Protein
Assay, Bio-Rad Laboratories, Hercules, CA) by comparison with
bovine serum albumin (BSA) standards. Expression of the following
cytokines and chemokines (IL-1b, IL-10, IL-12 [p40], IL-13, IL-18,
IFN-g, KC, MIP-1a, MIP-1b, MIP-2, MCP-1, and RANTES) was
determined using the Bioplex assay system (Bio-Rad Laboratories).
Cell Culture Experiments
To confirm the effects of epithelial Nrp1 deletion in vivo, in vitro
experiments were performed in A549 cells, a type II alveolar cell line.
Cells were purchased from ATCC (Manassus, VA), and maintained
according to the manufacturer’s instructions. Nrp1 silencing was
achieved using siRNA (Dharmacon, Thermo Scientific, Lafayette,
CO) (see online supplement for further details).
Assessment of Apoptotic Cell Death In Vitro
The effects of Nrp1 silencing on cell death were determined after
treatment with 10% CS extract (CSE) (see the online supplement for
further details), or 10 mM ceramide 8:0 (Avanti Polar Lipids, Alabas-
ter, AL) for 24 hours. Results were normalized to cells undergoing
mock transfection and vehicle treatment (fetal bovine serum–free
medium for CSE, or 0.1% ethanol for ceramide) for the same duration
and compared with cells transfected with nontargeting siRNA. Both
CSE and ceramide were previously reported to produce endothelial
and alveolar septal cell apoptosis and airspace enlargement in rodent
models (44, 46) as well as endothelial and epithelial cell death in vitro
(47–50). Apoptosis was quantified using the Apopercentage kit (Bio-
color Ltd., Accurate Chemical and Scientific Corporation, Westbury,
NY) following the manufacturer’s instructions. Quantification of re-
leased intracellular dye was performed using a microplate fluorimeter.
In addition, apoptotic nucleosomal release in cell lysates was measured
using the Cell Death ELISAPLUSkit (Roche Diagnostics, Indianapolis,
IN). Pan-caspase inhibition was achieved using a 30-minute pretreat-
ment with 50 mM carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluo-
romethylketone (ZVAD-fmk; Promega).
Statistical analysis was performed using the StatView program (SAS
Institute, Cary, NC). For studies evaluating the effects of conditional
Nrp1 deletion in neonatal or adult mice on airspace size, comparisons
between groups were made using one-way analysis of variance
(ANOVA). The combined effects of conditional Nrp1 deletion and
CS exposure were evaluated by two-way ANOVA. Two-way ANOVA
was also used for comparisons between groups for in vitro studies
evaluating the effects of Nrp1 silencing in A549 cells. When significant
variance ratios were obtained, least significant differences were calcu-
lated to allow comparison of individual group means. P values of 0.05
or less were considered significant.
Effects of Pulmonary Epithelial Nrp1 Deletion in Neonatal and
Conditional deletion of alveolar epithelial Nrp1 was confirmed
by immunohistochemical staining (see Figure E2 in the online
supplement). Inducible deletion of pulmonary epithelial Nrp1
in either the postnatal period (Figure E3) or adulthood (Figure
1) caused a small but statistically significant increase in airspace
size. Representative morphology from each condition is shown
in the top panels (Figure E3A and Figure 1A), whereas average
MLI and MCL for all animals, assessed by computer-assisted
morphometry, is shown in the bottom panels (Figure E3B and
Figure 1B). As has been previously reported (51, 52), the
presence of the CCSP-rtTA transgene in littermates resulted
in airspace enlargement relative to control mice that were
CCSP-rtTA negative despite similar expression of Nrp1.
CCSP-rtTA1littermates were therefore used as controls for
subsequent experiments. However, Nrp1 deletion in CCSP-
rtTA/tetO-Cre/Nrp1flox/floxmice caused additional airspace en-
largement, which was demonstrated by significant increases in
MLI and MCL following doxycycline treatment, when compared
with both littermate controls and CCSP-rtTA/tetO-Cre/Nrp1flox/1
mice. No significant inflammatory infiltrates were noted by
histologic evaluation after conditional Nrp1 deletion in either
postnatal or adult animals. These data suggest that Nrp1 plays
a role in alveolar structural maintenance both in the postnatal
and adult mouse.
Effects of CS Exposure and Pulmonary Epithelial Nrp1
Deletion on Alveolar Structure
There is evidence that disruption of structural maintenance
programs in the lung may predispose animals to CS-induced
alveolar destruction (53, 54). Because we noted mild airspace
enlargement after conditional deletion of epithelial Nrp1 in
Nrp1 deletion in adult mice. (A) Representative sections (103) of lungs
from littermate controls (top panel), CCSP-rtTA/tetO-Cre/Nrp1flox/1
(bottom left) and CCSP-rtTA/tetO-Cre/Nrp1flox/flox(bottom right) mice
treated with doxycycline chow from 6 weeks of age for 12 weeks (Bar 5
100 mm). (B) Average (6 SE) mean linear intercept (MLI) and mean
chord length (MCL) increased significantly in CCSP-rtTA/tetO-Cre/
Nrp1flox/floxmice after 12 weeks of conditional Nrp1 deletion (n 5 6
mice/group). *P < 0.0l vs. littermates;
tetO-Cre/Nrp1flox/1; black bars 5 MLI; gray bars 5 MCL.
Lung morphometry after conditional pulmonary epithelial
fP < 0.01 vs. CCSP-rtTA/
398AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 180 2009
both neonatal and adult animals, we sought to determine whe-
ther conditional epithelial Nrp1 deletion exacerbated the in-
jurious effects of CS in the lung. Adult (8–9 wk of age) CCSP-
rtTA/tetO-Cre/Nrp1flox/floxmice and littermate controls were
used for these experiments. Mice were exposed to CS or FA
for 12 weeks. Doxycycline-containing chow was initiated 3 days
before CS or FA exposure and was maintained for the dura-
tion of the exposure. As shown in Figure 2, this duration and
dose of CS did not cause morphologic alterations in litter-
mates, which is consistent with previous reports that indicate
that up to 6 months of CS exposure may be required for the
development of an emphysematous phenotype (44, 55). In
contrast, loss of epithelial Nrp1 significantly enhanced the
development of airspace enlargement (MLI, 47.4 6 1.7 vs.
52.4 6 1.3; S:V ratio, 0.043 6 0.002 vs. 0.039 6 0.001; P , 0.05)
in response to CS exposure for 3 months. These data convinc-
ingly demonstrate that the effects of chronic CS exposure on
alveolar structure were potentiated after conditional epithelial
Effects of CS Exposure and Pulmonary Epithelial Nrp1
Deletion on Indices of Cell Proliferation and Death
Augmented apoptosis (39, 44, 46) and attenuated proliferation
(56, 57) of alveolar cells were shown to be critical for the
maintenance of normal alveolar structure in other animal
models of emphysema. Evidence suggests that high levels of
Nrp1 may attenuate apoptosis (58). We therefore tested
whether conditional Nrp1 deletion altered the balance between
CS-induced cell death and proliferation in our model, thereby
leading to altered alveolar structure. Cell proliferation was
assessed by immunostaining lungs for PCNA, and apoptosis
was determined by immunostaining for active caspase 3 and in
situ DNA fragmentation (TUNEL). As shown in Figure 3,
a trend for fewer PCNA positive nuclei was noted in CCSP-
rtTA/tetO-Cre/Nrp1flox/floxmice exposed to both FA and CS,
although these differences did not achieve statistical signifi-
cance. In contrast, as shown in Figure 4, pulmonary epithelial
Nrp1 deletion caused increased CS-induced apoptosis, which
was demonstrated by expression of active caspase 3. To de-
termine the specific cell types undergoing apoptotic cell death,
TUNEL staining was performed with colocalization for type I
and II alveolar epithelial cell antigens and sections were
analyzed using confocal microscopy. CS exposure markedly
enhanced apoptosis of both type I and II alveolar epithelial cells
in lungs from CCSP-rtTA/tetO-Cre/Nrp1flox/floxmice, as shown
in Figure 5.
Figure 2. Lung morphometry after cigarette smoke (CS) exposure and
conditional pulmonary epithelial Nrp1 deletion. (A) Representative
histology (103) from CCSP-rtTA/tetO-Cre/Nrp1flox/floxmice (bottom
panels) and littermates (top panels) after conditional pulmonary epi-
thelial Nrp1 deletion and simultaneous CS or filtered air (FA) exposure
for 12 weeks. Prominent intraalveolar macrophages in CS-exposed
Nrp1–deficient mice are highlighted (insert, lower right panel) (Bar 5
100 mm). (B) Average (6 SE), mean linear intercept (MLI) increased and
surface-to-volume ratio (S:V) decreased after simultaneous pulmonary
epithelial Nrp1 deletion and CS exposure (n 5 8–9 mice per group).
White bars 5 CCSP-rtTA/tetO-Cre/Nrp1flox/flox; black bars 5 littermates.
after cigarette smoke (CS) exposure and pulmonary epithelial Nrp1
deletion. (A) Representative PCNA (green fluorescence; left panel) and
nuclear 49-69-diamidino-2-phenylindole (DAPI) (blue fluorescence; middle
panel), with merged image (right panel), following CS exposure and
conditional Nrp1 deletion (603 magnification). (B) Relative expression
(mean 6 SE) of PCNA-positive nuclei, normalized to total cell number
(assessed by nuclear staining with DAPI). Data were averaged from 10
random fields per mouse, and a minimum of 500 nuclei were counted for
each animal. There tended to be fewer PCNA positive cells in CCSP-rtTA/
tetO-Cre/Nrp1flox/floxmice compared with littermate controls, but these
differences did not achieve statistical significance. Relative expression of
PCNA was not altered by CS exposure in mice of either genotype. White
bars 5 CCSP-rtTA/tetO-Cre/Nrp1flox/flox; black bars 5 littermates.
Proliferating cell nuclear antigen (PCNA) immunostaining
Le, Zielinski, He, et al.: Neuropilin-1 and Emphysema 399
Effects of CS Exposure and Pulmonary Epithelial Nrp1
Deletion on VEGF Expression and Signaling
Because VEGF, a Nrp1 ligand, promotes cell survival (38, 59)
and acts as a mitogen for both epithelial (60–62) and endothelial
(63) cells, we measured the concentration of VEGF in both lung
lavage and tissue lysates. As indicated in Figure 6 (top panel),
levels of VEGF in lung lavage fluid did not differ significantly
between conditionally Nrp1–deficient mice and littermates
maintained in FA. However, although lavage VEGF concen-
tration increased significantly after CS exposure in littermate
control mice, this effect was lost in CS-exposed CCSPrtTA/
tetOCre/Nrp1flox/floxanimals (P < 0.05). Similar trends in
VEGF protein concentration were seen in lung tissue lysates,
although lysate differences between mice did not achieve
statistical significance (Figure 6, bottom panel). We also assayed
expression of phosphorylated and total VEGFR2 in whole lung
lysates, as Nrp1 has been shown to alter VEGF signaling pri-
marily via VEGFR2, and pharmacologic inhibition of VEGFR2
has been shown to promote apoptosis and airspace enlargement
in rodents. As shown in Figure 7, expression of phosphorylated
VEGFR2 was not significantly altered by tissue selective con-
ditional deletion of Nrp1 or CS, and similarly, total VEGFR2
Figure 4. Immunostaining for cleaved caspase 3 after cigarette smoke
(CS) exposure and pulmonary epithelial Nrp1 deletion. (A) Represen-
tative sections (603) of lungs from littermate controls (top panels), and
CCSP-rtTA/tetO-Cre/Nrp1flox/floxmice (bottom panels) after simulta-
neous CS or filtered air (FA) exposure and doxycycline-induced
pulmonary epithelial Nrp1 deletion for 12 weeks. Green fluorescence
demonstrates cytoplasmic staining for cleaved caspase 3 (white arrows).
The number of cells expressing cleaved caspase 3 was normalized to
total cell number estimated by nuclear staining with 49-69-diamidino-2-
phenylindole (DAPI) (blue fluorescence). (B) Relative expression (mean
6 SE) of cleaved caspase 3, normalized to DAPI, increased following CS
exposure in CCSP-rtTA/tetO-Cre/Nrp1flox/floxmice. Data were averaged
from 10 random fields per mouse, and a minimum of 500 nuclei were
counted for each animal. White bars 5 CCSP-rtTA/tetO-Cre/Nrp1flox/flox;
black bars 5 littermates; *P , 0.05.
exposure and conditional pulmonary epithelial Nrp1 deletion. (A)
Representative images obtained using confocal microscopy following
terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
staining (white fluorescence), and coimmunostaining for type I (T1-a;
red fluorescence) and type II (SP-C; green fluorescence) alveolar epithe-
lial cells. TUNEL-positive type I cells are marked by yellow arrows, and
type II cells by white arrows. DNA fragmentation following CS exposure
was seen in both type I and II alveolar epithelial cells. TUNEL positive
cells were quantified after nuclear staining 49-69-diamidino-2-phenyl-
indole (DAPI) (blue fluorescence). Five random fields were captured for
each animal. Top panels 5 littermates; bottom panels 5 CCSP-rtTA/
tetO-Cre/Nrp1flox/flox. (B) Relative expression (mean 6 SD) of TUNEL
positive type I and type II alveolar epithelial cells increased following CS
exposure in CCSP-rtTA/tetO-Cre/Nrp1flox/floxmice. Data were normal-
ized to values in littermate controls exposed to filtered air (FA). Black
bars 5 FA-exposed littermates; gray bars 5 CS-exposed littermates;
white bars 5 FA-exposed CCSP-rtTA/tetO-Cre/Nrp1flox/flox; striped bars 5
CS-exposed CCSP-rtTA/tetO-Cre/Nrp1flox/flox; *P , 0.05.
Increased DNA fragmentation after cigarette smoke (CS)
400 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 1802009
expression did not differ significantly between CCSP-rtTA/
tetO-Cre/Nrp1flox/floxmice and littermate controls after FA or
CS exposure. As expected, under control (FA) conditions,
expression of Nrp1 protein was decreased in the lung lysates
from CCSP-rtTA/tetO-Cre/Nrp1flox/floxmice when compared with
littermates. Also shown in Figure 7, Nrp1 expression decreased in
control mice after chronic CS exposure to levels similar to those
seen with conditional Nrp1 deficiency. These data suggest that
conditional deletion of Nrp1 attenuated CS-induced increases in
lung lavage VEGF protein concentration without significantly
reducing VEGFR2 expression or phoshorylation.
Effects of CS Exposure and Pulmonary Epithelial Nrp1
Deletion on Inflammation and Inflammatory
See the online supplement for further details.
Effects of Nrp1 Silencing on Epithelial Cell Apoptosis In Vitro
To confirm a role for epithelial Nrp1 deletion in the augmenta-
tion of cell death, we used siRNA to silence Nrp1 in A549 cells,
a human alveolar type II cell line, and then evaluated the
susceptibility of these cells to apoptosis. As shown in Figure 8,
the silencing of Nrp1 resulted in enhanced apoptosis after
exposure of A549 cells to 10% CSE (top panel) or ceramide
(bottom panel) for 24 hours. In contrast, apoptosis of vehicle-
treated cells was not altered by transfection with Nrp1 siRNA in
either series of experiments. Enhanced cell death in response
to both CSE and ceramide was significantly attenuated by
pretreatment with the pan-caspase inhibitor ZVAD-fmk (Figure
9). Taken together, these data support a critical role for Nrp1 in
alveolar epithelial cell survival following exposure to stimuli
relevant to emphysema pathophysiology.
Cigarette smoke is the single most prevalent risk factor for the
development of COPD, yet not all smokers develop this disorder
(5, 6). Several hypotheses have evolved in an attempt to un-
derstand how CS causes alveolar destruction and emphysema,
and particularly why certain hosts exhibit increased susceptibility
to the injurious effects of CS exposure. For some time, inves-
tigators have focused on inflammation and protease/antiprotease
imbalance as the mechanism underlying emphysema develop-
Figure 7. Effects of conditional pulmonary epithelial Nrp1 deletion and
cigarette smoke (CS) exposure on expression of Nrp1, p-vascular
endothelial growth factor (VEGF)R2, and VEGFR2 in lung lysates.
Representative expression of Nrp1, p-VEGFR2, and VEGFR2 by Western
blot analysis of lung lysates (top panel), and mean (6 SE) changes in
expression, assessed by densitometry (lower panel; n 5 4–6/group).
Expression of Nrp1 was significantly lower in lung lysate from CCSP-
rtTA/tetO-Cre/Nrp1flox/floxmice after filtered air (FA) or chronic CS
when compared with FA-exposed littermates. Exposure of littermate
control mice to chronic CS also resulted in decreased Nrp1 expression
when compared with FA. There were no significant differences detected
in expression of either p-VEGFR2 or total VEGFR2 as a result of conditional
pulmonary epithelial Nrp1 deletion, or chronic CS exposure in either
littermate controls or CCSP-rtTA/tetO-Cre/Nrp1flox/floxmice. 1 5 FA,
littermate; 2 5 FA, CCSP-rtTA/tetO-Cre/Nrp1flox/flox; 3 5 CS, littermate;
4 5 CS, CCSP-rtTA/tetO-Cre/Nrp1flox/flox. White bars 5 CCSP-rtTA/tetO-
Cre/Nrp1flox/flox; black bars 5 littermates; *P , 0.05.
pulmonary epithelial Nrp1 deletion on vascular endothelial growth
factor (VEGF) concentration. VEGF concentration (mean 6 SE), mea-
sured using ELISA, in lung lavage fluid (top panel; n 5 4–5/group) and
lung tissue homogenates (bottom panel; n 5 6–7/group). In littermate
control mice, VEGF concentration increased significantly after CS
exposure compared with filtered air (FA). In contrast, no CS-induced
increase in VEGF concentration was seen in Nrp1–deficient mice. A
similar trend was seen when VEGF concentration was measured in lung
tissue homogenate, although these differences did not reach statistical
significance. Top panel, *P , 0.05 vs. littermates; bottom panel,fP ,
0.05 vs. FA. White bars 5 CCSP-rtTA/tetO-Cre/Nrp1flox/flox; black bars 5
Effects of cigarette smoke (CS) exposure and conditional
Le, Zielinski, He, et al.: Neuropilin-1 and Emphysema 401
ment, and ample clinical and experimental evidence supports this
concept (64, 65). Although CS causes enhanced lung inflamma-
tion with the potential for an imbalance of protease/antiprotease
activity, it remains unclear why emphysema develops only after
decades of smoking. Studies using knockout mice have shown
that extracellular matrix proteases and inflammatory cells
are required for CS-induced emphysema, which in the mouse
develops after 6 months of ongoing exposure (44, 65, 66).
Because mounting evidence suggests that the apoptotic
machinery is also necessary to generate an emphysema pheno-
type (64, 67–69), an alternative and potentially complementary
hypothesis to explain host susceptibility to the injurious effects
of chronic smoke exposure has evolved. This hypothesis sug-
gests that CS-induced alveolar destruction could be exacerbated
by abnormalities in pathways critical for lung development and
postnatal lung homeostasis, creating an imbalance between
cellular programs regulating lung injury and repair (69, 70).
Several lines of investigation have supported a role for the
disruption of alveolar structural maintenance in the pathogenesis
of emphysema. Published literature describes spontaneous air-
space enlargement in mice that are conditionally deficient for
VEGF (71) or components of other growth factor signaling
cascades that are active during normal lung development (72–
76). Additional studies demonstrate that dysregulation of pro-
grammed cell death in the adult lung may lead to airspace
enlargement in the absence of marked inflammation (39, 46, 77,
78). It was also shown that alteration of genes associated with
cellular senescence, a state characterized by impaired recovery
from injury, may sensitize mice to CS-induced inflammation and
emphysema (54). These data suggest that inflammation alone
cannot mediate the destructive elements of cigarette smoke in
ceramide-induced apoptosis of A549 cells. Exposure to either 10% CSE
(n 5 4/condition; top panel, gray bars 5 10% CSE; black bars 5 10%
CSE 1 ZVAD; white bars 5 vehicle 1 ZVAD), or 10 mM ceramide 8:0
(n 5 8 /condition; middle panel, gray bars 5 10% ceramide 8:0; black
bars 5 ceramide 8:0 1 ZVAD; white bars 5 vehicle 1 ZVAD) for 24 hours
resulted in apoptosis, which was augmented by silencing of Nrp1 and
attenuated by pretreatment of cells for 30 minutes with the pan-
caspase inhibitor ZVAD-fmk (50 mM). Cell death was assessed using the
apopercentage assay (top and middle panels), and results were normal-
ized to vehicle-treated, mock-transfected cells. *P , 0.05 vs. vehicle 1
ZVAD, CSE 1 ZVAD;fP , 0.05 vs. NT siRNA (top panel). *P , 0.05 vs.
vehicle 1 ZVAD, ceramide 1 ZVAD;fP , 0.05 vs. NT siRNA (middle
panel). To confirm that Nrp1 silencing enhanced CSE-induced apopto-
sis, we also evaluated cell death after exposure to 10% CSE for 24 hours
by measurement of nucleosome release in cell lysates using the Cell
Death ELISAPLUSkit (n 5 8/condition; bottom panel, gray bars 5 10%
CSE; black bars 5 10% CSE 1 ZVAD; white bars 5 vehicle 1 ZVAD).
Results for this assay were normalized to vehicle-treated, mock-trans-
fected cells. Results shown are mean 6 SE. *P , 0.05 vs. vehicle 1
ZVAD, CSE 1 ZVAD;fP , 0.05 vs. NT siRNA (bottom panel).
Effects of ZVAD-fmk on cigarette smoke extract (CSE)- or
extract (CSE)- and (bottom panel) ceramide-induced apoptosis of A549
cells. Apoptosis of A549 cells following 24 hours of exposure to CSE
(10%; n 5 6/condition; top panel) or ceramide 8:0 (10 mM; n 5 5/
condition, bottom panel) was augmented after silencing of Nrp1.
Shown for comparison are results in A549 cells transfected with
nontargeting (NT) siRNA. In contrast, silencing of Nrp1 had no effect
on apoptosis (white bars, both panels) of vehicle-treated cells for either
series of experiments. Cell death was assessed using the Apopercentage
assay, and results were normalized to vehicle treated, mock-transfected
cells. Results shown are mean 6 SE. *P , 0.05 vs. vehicle;fP , 0.05 vs.
NT siRNA. Top panel, white bars 5 vehicle; gray bars 5 10% CSE. Bottom
panel, white bars 5 vehicle; black bars 5 ceramide 10 mM.
Effects of Nrp1 silencing on (top panel) cigarette smoke
402AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 1802009
the lung or that alveolar destruction is triggered by distinct
chronic functional and molecular signatures in the inflammatory
processes. The present work proposes that disruption of alveolar
maintenance signals linked to epithelial Nrp1 signaling exacer-
bates alveolar injury provoked by CS, leading to airspace
Although several groups have described expression of Nrp1
in alveolar epithelium (24, 25), little is known regarding its
function in these cells. VEGF has autocrine effects on growth
and proliferation of pulmonary epithelial cells in vitro and may
protect against oxidant-induced apoptosis of cultured primary
lung epithelial cells and epithelial-derived cell lines (38, 60).
Moreover, VEGF stimulates branching morphogenesis of
renal tubular epithelium in culture (61). Whether Nrp1 is
required for these effects of VEGF on epithelial function is
unknown. Furthermore, Sema3A, the alternate ligand for
Nrp1, was shown to inhibit alveolar septation in cultured fetal
lung explants in a Nrp1–dependent manner (24), raising the
possibility that disruption of the Sema3A–Nrp1 signaling axis
may be an important determinant of airspace size. Because
Nrp1 is highly expressed in pulmonary epithelium and is an
integral component of the cellular receptor complexes in-
volved in both alveolar septation and vascular development,
we sought to determine if epithelial Nrp1 was required for the
maintenance of normal alveolar structure and/or altered sus-
ceptibility to CS-induced emphysema. Our data from filtered
air-exposed mice demonstrate mild airspace enlargement after
deletion of pulmonary epithelial Nrp1 in both postnatal and
adult animals compared with littermate controls. One poten-
tial limitation of this model is that our data in Nrp1-suffi-
cient CSSP-rtTA1littermates corroborate previous reports of
nonspecific effects of the CCSP-rtTA transgene on lung
morphometry (51, 52). However, our studies revealed that
pulmonary epithelial-specific Nrp1 deletion has additional
effects on morphometric measures of airspace size. We there-
fore believe that our data support a role for epithelial Nrp1 in
the development and maintenance of normal adult alveolar
More striking than the effects of conditional pulmonary
epithelial Nrp1 deletion in the FA-exposed neonatal or adult
lung were the effects of chronic CS exposure in conditionally
Nrp1–deficient mice. A potential role for Nrp1 in CS-induced
emphysema is supported by recent data demonstrating reduced
expression of Nrp1 protein in the lungs of smokers with COPD
compared with lungs from smokers who had normal lung
function as well as nonsmoking controls (40). Consistent with
this published data in human lungs, our experiments demon-
strate decreased Nrp1 expression in tissue lysate from control
mice exposed to CS for 3 months compared with those exposed
to air. Importantly, however, we found that airspace enlarge-
ment in response to chronic smoke was significantly amplified in
mice after pulmonary epithelial Nrp1 deletion.
Because Nrp1 modulates the balance between cell prolifer-
ation and survival in other cell types (16, 17, 19, 58), we
compared indices of proliferation and programmed cell death
in the lungs of mice after FA and CS exposure. Overall, there
tended to be fewer proliferating cells in the distal lung
parenchyma of CCSP-rtTA/tetO-Cre/Nrp1flox/floxmice than in
littermate controls, but these differences were not statistically
significant, and the amount of cell proliferation was not altered
by chronic smoke exposure in mice of either genotype. In
contrast, apoptosis was markedly enhanced in conditionally
Nrp1–deficient mice exposed to CS compared with FA controls
and CS-exposed littermates. Our in vivo colocalization data
suggest that Nrp1 deletion enhanced the susceptibility of both
type I and type II alveolar epithelial cells to CS-induced
programmed cell death. Furthermore, in vitro experiments
confirmed that Nrp1 silencing significantly augmented apoptosis
after acute exposure of A549 cells to either CSE or ceramide (a
proposed mediator of airspace remodeling in emphysema ),
whereas reduction of Nrp1 using RNA interference did not
enhance cell death in vehicle-treated cells. Taken together,
these findings suggest that Nrp1 modulates alveolar epithelial
cell survival after exposure to oxidative stressors.
Because disruption of VEGFR2 signaling has been shown
to enhance alveolar cell apoptosis and alveolar destruction (39,
46, 77), one could speculate that the effects of pulmonary
epithelial Nrp1 deletion are secondary to altered VEGF signal
transduction. Our data suggest that lavage VEGF concentra-
tion increased in response to CS in littermate control mice.
This finding is consistent with the study of Wright and
colleagues (79), demonstrating time-dependent up-regulation
of VEGF expression from the lungs of smoke-exposed guinea
pigs. In contrast, chronic CS exposure had no effect on VEGF
concentration in lavage from conditionally Nrp1 deficient
mice. Higher VEGF concentrations after smoke exposure
might be predicted to enhance cellular proliferation, or limit
apoptosis, in response to injury. It is therefore reasonable to
pose a causal link between lower VEGF concentration and
increased apoptosis in the alveolar epithelium. However, the
effects of VEGF on cell proliferation and survival (15, 30, 39,
60, 61) and airspace enlargement (39, 46, 77) in previous
studies were primarily mediated through VEGFR2, and tissue
levels of phosphorylated VEGFR2 were not significantly de-
creased after chronic smoke exposure and conditional epithe-
lial Nrp1 deletion. Although VEGFR2 protein expression
measured in whole lung lysates may not reflect changes in
a specific cell compartment, this suggests that changes in
lavage VEGF concentration may not explain differences
in alveolar epithelial cell death. Because epithelial cells are
the primary source of VEGF in lung lavage, an alternative
explanation linking conditional epithelial Nrp1 deletion and
decreased lavage VEGF concentrations in CS-exposed CCSP-
rtTA/tetO-Cre/Nrp1flox/floxmice could be that enhanced epi-
thelial cell death decreases VEGF production. Furthermore,
although several investigators have demonstrated that the
effects of Nrp1 on cell proliferation and death may depend
on how this receptor modulates the balance between VEGF
and Sema3 signaling (17, 26), other studies also demonstrate
that Nrp1 may alter cell survival independent of its effects on
VEGFR activation (22, 80–82).
Lung lavage inflammatory cell type and number were not
significantly altered by conditional pulmonary epithelial Nrp1
deletion, although histology suggested a possible mild macro-
phage accumulation in alveoli. Differences in smoke-induced
MMP activity and inflammatory mediator production that
resulted from conditional Nrp1 deficiency were subtle. It is
possible that alterations in alveolar maintenance due to disrup-
tion of Nrp1 signaling leads to secondary, mild, smoke-induced
inflammation, which could play a contributory role to alveolar
destruction in this model. Alternatively, it is known that
phagocytosis of apoptotic cells by tissue macrophages is asso-
ciated with minimal proinflammatory cytokine release. A
possible explanation for the presence of macrophage infiltration
in CS-exposed Nrp1-deficient mice, but not in CS-exposed
littermates, would be activation, or possible dysregulation, of
normal apoptotic clearance pathways (83, 84).
In summary, our data support a role for pulmonary epithelial
Nrp1 in the maintenance of normal alveolar structure. Condi-
tional deletion of Nrp1 in lung epithelial cells exacerbated the
injurious effects of chronic CS exposure, promoting the de-
velopment of apoptosis of Type I and Type II alveolar epithelial
Le, Zielinski, He, et al.: Neuropilin-1 and Emphysema 403
cells, and airspace enlargement. In vitro experiments confirmed
that loss of Nrp1 augmented alveolar epithelial cell death
following acute exposure to cigarette smoke extract and short
chain ceramides. Overall, these data support the hypothesis that
dysregulation of key developmental signal transduction path-
ways in the adult lung may alter the balance between cell injury
and repair in response to toxic exposures such as cigarette
smoke, thereby accelerating airspace enlargement and the de-
velopment of emphysema.
Conflict of Interest Statement: A.L. does not have a financial relationship with
a commercial entity that has an interest in the subject of this manuscript. R.Z.
does not have a financial relationship with a commercial entity that has an
interest in the subject of this manuscript. C.H. does not have a financial
relationship with a commercial entity that has an interest in the subject of this
manuscript. M.T.C. does not have a financial relationship with a commercial
entity that has an interest in the subject of this manuscript. S.B. received $5,001
to $1,000 in consultancy fees from Merck; up to $1,000 in lecture fees from
Novartis; and more than $100,000 in industry-sponsored grants from Quark
Pharmaceuticals. R.M.T. does not have a financial relationship with a commercial
entity that has an interest in the subject of this manuscript. P.M.B. does not have
a financial relationship with a commercial entity that has an interest in the subject
of this manuscript.
Acknowledgment: The authors thank Robin Yachechko and Ozlem Gurkan for
expert technical assistance; Jeffrey Whitsett and Mary Williams for providing
critical reagents; Robert Wise, Elizabeth Wagner, and Rachel Damico for their
critical reading of the manuscript; and David Ginty for his generous support with
both reagents and scientific guidance.
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