Prenatal inflammation exacerbates hyperoxia-induced functional and structural changes in adult mice

ArticleinAJP Regulatory Integrative and Comparative Physiology 303(3):R279-90 · June 2012with14 Reads
Impact Factor: 3.11 · DOI: 10.1152/ajpregu.00029.2012 · Source: PubMed
Abstract

Maternally derived inflammatory mediators, such as IL-6 and IL-8, contribute to preterm delivery, low birth weight, and respiratory insufficiency, which are routinely treated with oxygen. Premature infants are at risk for developing adult-onset cardiac, metabolic, and pulmonary diseases. Long-term pulmonary consequences of perinatal inflammation are unclear. We tested the hypothesis that a hostile perinatal environment induces profibrotic pathways resulting in pulmonary fibrosis, including persistently altered lung structure and function. Pregnant C3H/HeN mice injected with LPS or saline on embryonic day 16. Offspring were placed in room air (RA) or 85% O(2) for 14 days and then returned to RA. Pulmonary function tests, microCTs, molecular and histological analyses were performed between embryonic day 18 and 8 wk. Alveolarization was most compromised in LPS/O(2)-exposed offspring. Collagen staining and protein levels were increased, and static compliance was decreased only in LPS/O(2)-exposed mice. Three-dimensional microCT reconstruction and quantification revealed increased tissue densities only in LPS/O(2) mice. Diffuse interstitial fibrosis was associated with decreased micro-RNA-29, increased transforming growth factor-β expression, and phosphorylation of Smad2 during embryonic or early fetal lung development. Systemic maternal LPS administration in combination with neonatal hyperoxic exposure induces activation of profibrotic pathways, impaired alveolarization, and diminished lung function that are associated with prenatal and postnatal suppression of miR-29 expression.

Full-text

Available from: Stephen E Welty, May 03, 2016
Prenatal inflammation exacerbates hyperoxia-induced functional and structural
changes in adult mice
Markus Velten,
1,2
Rodney D. Britt Jr.,
1
Kathryn M. Heyob,
1
Stephen E. Welty,
3
Britta Eiberger,
4
Trent E. Tipple,
1
and Lynette K. Rogers
1
1
Center for Perinatal Research, The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio;
2
Department
of Anesthesiology and Intensive Care Medicine, Rheinische Friedrich-Wilhlems-University, University Medical Center, Bonn,
Germany;
3
Texas Children’s Hospital, Baylor College of Medicine, Houston, Texas; and
4
Institute of Anatomy, Rheinische
Friedrich-Wilhlems-University, Bonn, Germany
Submitted 20 January 2012; accepted in final form 9 June 2012
Velten M, Britt Jr. RD, Heyob KM, Welty SE, Eiberger B,
Tipple TE, Rogers LK. Prenatal inflammation exacerbates hyperoxia
induced functional and structural changes in adult mice. Am J Physiol
Regul Integr Comp Physiol 303: R279 –R290, 2012. First published
June 20, 2012; doi:10.1152/ajpregu.00029.2012.—Maternally derived
inflammatory mediators, such as IL-6 and IL-8, contribute to preterm
delivery, low birth weight, and respiratory insufficiency, which are
routinely treated with oxygen. Premature infants are at risk for
developing adult-onset cardiac, metabolic, and pulmonary diseases.
Long-term pulmonary consequences of perinatal inflammation are
unclear. We tested the hypothesis that a hostile perinatal environment
induces profibrotic pathways resulting in pulmonary fibrosis, includ-
ing persistently altered lung structure and function. Pregnant C3H/
HeN mice injected with LPS or saline on embryonic day 16. Offspring
were placed in room air (RA) or 85% O
2
for 14 days and then returned
to RA. Pulmonary function tests, microCTs, molecular and histological
analyses were performed between embryonic day 18 and 8 wk. Alveo-
larization was most compromised in LPS/O
2
-exposed offspring. Collagen
staining and protein levels were increased, and static compliance was
decreased only in LPS/O
2
-exposed mice. Three-dimensional microCT
reconstruction and quantification revealed increased tissue densities only
in LPS/O
2
mice. Diffuse interstitial fibrosis was associated with de-
creased micro-RNA-29, increased transforming growth factor- ex-
pression, and phosphorylation of Smad2 during embryonic or early
fetal lung development. Systemic maternal LPS administration in
combination with neonatal hyperoxic exposure induces activation of
profibrotic pathways, impaired alveolarization, and diminished lung
function that are associated with prenatal and postnatal suppression of
miR-29 expression.
pulmonary fibrosis; transforming growth factor-; fetal origins; mi-
crocomputed tomography scans; pulmonary function tests
THE CAUSES AND CONSEQUENCES of preterm birth remain poorly
understood and present a significant health burden. In the past
three decades, advances in neonatal care, including use of
antenatal corticosteroids, surfactant therapy, and high-fre-
quency ventilation, have significantly improved survival rates
of extremely preterm (28 wk gestation) and low-birthweight
infants (45). However, little is known about the long-term
physiological consequences of a hostile perinatal environment.
Data currently being collected indicate that preterm infants
surviving to adulthood are at greater risk for the development
of chronic health problems (9 –11). Low birth weight, early
gestational age, and respiratory support are highly associated
with interrupted alveolarization and respiratory insufficiency.
These pathologies can progress into the development of bron-
chopulmonary dysplasia (BPD) (29), a disease pathologically
characterized by impaired alveolarization and diffuse intersti-
tial fibrosis (2, 26). Furthermore, whether they develop BPD or
not, extremely immature infants are at increased risk for
developing adult pulmonary pathologies, including emphy-
sema, chronic obstructive pulmonary disease, asthma, or pul-
monary fibrosis (2, 14, 15, 26, 36, 57).
Maternal infections and/or inflammation and the subsequent
inflammatory responses that contribute to preterm delivery can
significantly impact fetal development (16, 18, 19, 22, 58).
Research supporting the “fetal origins of adult disease” hy-
pothesis has focused on cardiovascular and metabolic diseases
(3, 4, 32, 43); however, disordered fetal development has
profound effects on other organs, including the lung. Recently,
Shi and colleagues (47, 48, 56) demonstrated that early expo-
sures during periods of developmental plasticity contribute to
the development of adult pulmonary diseases.
While the mechanisms responsible for the development of
pulmonary pathologies are multifactorial, common diseases are
often characterized by diminished lung function and interstitial
fibrosis. Mechanistically, lung fibrosis is associated with dys-
regulated transforming growth factor- (TGF-) expression
and Smad signaling in human patients and bleomycin-treated
mice (6, 7). Previous studies have implicated a crucial temporal
window for TGF- signaling during lung development, that, if
interrupted, leads to impaired alveolarization and pulmonary
fibrosis (47, 49). TGF- modulates the expression of profi-
brotic genes through suppression of micro-RNA (miR)-29 that,
in turn, causes increases in TGF- expression, in a feed-
forward manner (12, 38). miR-29 has been demonstrated to
target proteins regulated by TGF- and Smad signaling, such
as collagen and matrix-remodeling proteins (12). miRs regulate
the expression of multiple genes by enhancement, suppression,
or destabilization of target RNAs and are increasingly recog-
nized as important contributors to developmental processes and
disease pathogenesis. Furthermore, dysregulation of miRs has
been linked to fibrosis in multiple organs, including the heart
(52), kidney (8), and lung (12). The contributions of maternal
influences on early disruption of fetal TGF- pathways or miR
expressions, in developing lungs are unknown.
Many animal models that include fetal inflammation or
postnatal hypo- or hyperoxic exposures have been developed to
study newborn lung diseases. Hyperoxic exposure induces
inflammation and disrupts cell proliferation, leading to alveolar
Address for reprint requests and other correspondence: M. Velten, Dept. of
Anesthesiology and Intensive Care Medicine, Rheinische Friedrich-Wilhelms-
Univ., Univ. Medical Ctr., Sigmund-Freud-Str. 25, 53105 Bonn, Germany
(e-mail: Markus.Velten@UKB.uni-bonn.de).
Am J Physiol Regul Integr Comp Physiol 303: R279 –R290, 2012.
First published June 20, 2012; doi:10.1152/ajpregu.00029.2012.
0363-6119/12 Copyright
©
2012 the American Physiological Societyhttp://www.ajpregu.org R279
Page 1
dysplasia in newborn rodents (27, 60). However, the long-term
pulmonary consequences of fetal exposures have not been
extensively investigated (30). We have previously reported
developmental alterations in alveolarization and pulmonary
function 14 days after systemic maternal inflammation and
neonatal hyperoxic exposure (53). This model was designed to
mimic the hostile perinatal inflammatory environment often
encountered by prematurely born human infants. In the current
studies, we tested the hypothesis that the combination of
LPS-induced systemic maternal inflammation and postnatal
hyperoxic exposure would result in 1) persistently altered
alveolarization, 2) lung fibrosis, and 3) impaired pulmonary
function in adulthood.
MATERIALS AND METHODS
Animals and exposure. Animal study protocols were approved by
the Institutional Animal Care and Use Committee at The Research
Institute at Nationwide Children’s Hospital, Columbus, OH. All
animals were handled in accordance with National Institutes of Health
guidelines and housed in a “specified pathogen-free” facility. Mice
were housed in our facility at least 7 days before breeding was started,
and pregnancy was time dated by the presence of a vaginal plug.
Pregnant C3H/HeN mice were injected on embryonic day 16 (E16)
with LPS (80 g/kg ip, serotype 0111:B4, no. 437627; Calbiochem,
Gibbstown, NJ) or an equal volume of saline. The amount of LPS was
chosen on the basis of preliminary studies to determine the highest
dose that resulted in viable litter of equal size. Each litter of newborn
mice was paired with a litter born to a dam receiving the same E16
treatment, and the pups were pooled and redistributed randomly, as
previously described (53). One of the paired group of pups was
exposed to 85% O
2
for 2 wk (saline/O
2,
LPS/O
2
) and subsequently
returned to room air (RA), while the corresponding group was
maintained in RA (saline/RA, LPS/RA). Nursing dams were rotated
between their RA and O
2
litter every 24 h to prevent oxygen toxicity.
Twenty-four hours of RA or oxygen exposure was designated as day
1. The mice were killed at E18, 7 or 14 days, or 8 wk of life, and only
one pup per litter was used at each time point for analyses. One pup
per litter was analyzed per experiment, and equal numbers of males
and females were measured. For the pulmonary function tests, one
male and one female were analyzed from each litter.
Histology. The left lung was inflation fixed with 10% buffered
formalin at a pressure of 25 cm H
2
O for 15 min. Following paraffin
embedding, the tissue sections were cut, and 4-m slides were stained
with hematoxylin and eosin (H&E) for morphometric measurements,
Mason’s trichrome, and Picrosirius red (PSR) stain to assess collagen
deposition.
Immunohistochemistry. Inflation-fixed left lung tissue sections
were cut and 4-m slides stained for macrophages with Mac3 mono-
clonal antibody (catalog no. 550292, BD Pharmingen, San Diego,
CA) as the primary antibody and rabbit anti-rat (catalog no. BA-4001;
Vector, Burlingame, CA) as a secondary antibody. Macrophage counts
were performed on five nonoverlapping fields per mouse lung tissues and
n 5 animals per group using digital image analysis software with
settings for color and size identification (Image Pro Plus 4.0; Media
Cybernetics, Silver Spring, MD).
Western immunoblotting. Proteins were separated on SDS-PAGE
gels and transferred to PVDF membranes. Membranes were probed
with antibodies to collagen I (ab292; Abcam, Cambridge, MA),
collagen III (EMD Millipore, 234189; Millipore, Billerica, MA),
p-Smad2 (no. 3108, Cell Signaling, Danvers, MA), and total Smad2/3
(no. 3102; Cell Signaling). Blots were developed using enhanced
chemiluminescence (ECL Western blotting detection, GE Healthcare,
Chalfont, Buckinghamshire, UK), and expression levels were quanti-
fied using ImageQuant software, version 5.0 (Molecular Dynamics,
Sunnydale, CA). The density of the band for the protein of interest
was normalized to the density of -actin protein (no. ab6276; Abcam).
Pulmonary function tests. A SCIREQ FlexiVent (FlexiVent, SCIREQ,
Montreal, Canada) ventilator was used to perform pulmonary function
analyses. Mice were anesthetized with ketamine (200 mg/kg ip) and
xylazine (20 mg/kg), tracheotomized with a 20-gauge cannula (BD
Intramedic, no. 427564; Becton Dickinson, Franklin Lakes, NJ), and
connected to the FlexiVent ventilator. The plane of anesthesia was
sufficient to prevent spontaneous breathing. The mice were ventilated
with a tidal volume of 10 ml/kg at a frequency of 350 breaths/min and
positive end-expiratory pressure of 2 cm H
2
O to achieve lung volume
similar to spontaneous breathing. Forced oscillation (0.5–19.6 Hz)
was applied for 8 s. Subsequently, dynamic pressure-volume maneu-
vers were performed stepwise, increasing airway pressure to 30 cm
H
2
O and then reversing the process. For each parameter, three
measurements were assessed and averaged. Measurements were ex-
cluded from analyses if they were disrupted by a spontaneous breath,
and a coefficient of determination of 0.98 was used as the lower limit
for each measurement.
MicroCT imaging. A General Electric Healthcare Xplore Locus
microCT (General Electric, London, Canada) was used for pulmonary
imaging. Mice were anesthetized with ketamine (20 mg/kg ip) and
xylazine (2 mg/kg ip). Respiratory gated microCT images were
acquired at inspiration, as previously described (17). Images were
reconstructed with a nominal isotropic voxel spacing of 90 m. Lung
density data obtained from microCTs were normalized to density
standards and converted to Hounsfield units (HU). Five representative
regions of interest were measured, and the average was considered as
the lung density of the individual mouse (40). After performing
microCT in vivo, mice were killed by ketamine (150 mg/kg)–xylazine
(15 mg/kg) overdose; then they were tracheotomized, lungs were air
inflated, and post-mortem high-resolution microCT were performed at
a constant inflation of 30 cm H
2
O. Three-dimensional images were
reconstructed with a nominal isotropic voxel spacing of 20 m and
evaluated by a radiologist blinded to group assignment.
Quantitative real-time PCR. Total RNA was isolated from frozen
lung tissue using an RNeasy mini kit (Qiagen, Valencia, CA). cDNA
was synthesized using a Maxima first-strand cDNA synthesis kit for
RT-qPCR (no. K1641; Thermo Scientific Fermentas, Glen Burnie,
MD). Quantitative real-time PCR was performed using Maxima
SYBR Green/ROX qPCR Master Mix (K0222; Thermo Scientific
Fermentas) and the Mastercycler epgradient Realplex real-time PCR
detection system (Eppendorf, Hamburg, Germany).
ELISA. Frozen lungs were homogenized, and protein concentra-
tions were determined by Bradford assay. TNF-, IL-6, keratino-
cyte-derived chemokine (KC), and monocyte chemoattractant pro-
tein-1 (MCP-1) levels were measured using ELISA (Duoset ELISA
kits; R&D Systems, Minneapolis, MN), according to the manufac-
turer’s protocols. Absorbances were determined spectrophometri-
cally using a Spectramax M2 Plate Reader (Molecular Devices,
Sunnyvale, CA).
MicroRNA analyses. miR fractions were isolated and enriched from
lungs with Qiagen RNeasy Mini Kits (Qiagen, Valencia, CA). Sub-
sequently, 100-ng enriched miR was reverse-transcribed with SA
Biosciences miR first-strand kit (SA Biosciences, Frederick, MD).
Real-time PCR was performed on an Mastercycler epgradient Real-
plex real-time PCR detection system (Eppendorf, Hamburg, Ger-
many) using RT
2
SYBR Green qPCR Mastermix with ROX and RT
2
miRNA qPCR assay primer sets (SA Biosciences).
Statistics. Analyses were performed using GraphPad PRISM 5
(GraphPad, La Jolla, CA). Data are expressed as means SE.
Statistical analyses were performed using a two-way ANOVA with
Bonferroni post hoc or Student’s t-test. P 0.05 was considered
statistically significant.
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RESULTS
Lung and body weights. Body weights (BW) were assessed
after birth and at 8 wk of age. BW was significantly lower in
pups born to LPS-injected dams compared with pups born to
saline-injected dams (means SE; 1.22 0.01 vs. 1.32
0.02 g, P 0.001). At 8 wk, BW was less in saline/O
2
and
even lower in LPS/O
2
-exposed mice compared with saline/RA
controls. Absolute right lung weights were not different; how-
ever, ratios of right lung weight to BW were significantly
greater in LPS/O
2
-exposed mice than in saline/RA controls.
Neither absolute nor relative liver weights were different and
sexes were equally distributed (Table 1).
Lung morphometric. Alveolarization was assessed at 8 wk of
age. Morphometric analyses of H&E-stained lung sections
(Fig. 1A) revealed alveolar numbers that were 30%-45% lower
in adult mice exposed to either LPS/RA or saline/O
2
and 60%
lower in animals exposed to LPS/O
2
than the saline/RA con
-
trols (Fig. 1B). Mean alveolar areas were 72% greater in mice
exposed to LPS/RA (9,002 693 m
2
), 110% greater in mice
exposed to saline/O
2
(10,631 1,465 m
2
), and 123% greater
Table 1. Body, right lung, and liver weights from 8-wk-old mice after systemic maternal LPS and neonatal hyperoxia
exposure
Saline/RA Saline/O
2
LPS/RA LPS/O
2
BW, g 23.75 0.58 21.82 0.71* 22.77 0.62 20.76 0.62*
Right lung weight, mg 85.88 0.40 86.66 0.35 90.00 0.31 88.82 0.21
Ratio of right lung to BW, mg/g 0.36 0.02 0.40 0.02 0.40 0.01 0.44 0.02*
Liver weight, mg 141.29 4.94 131.87 5.07 130.38 3.31 126.00 3.92
Ratio of liver weight to BW, mg/g 6.00 0.25 6.10 0.24 5.81 0.12 6.10 0.15
Values are expressed as means SE. Data were analyzed by one-way ANOVA followed by Bonferroni post hoc test. n 15–18 mice per group. *P 0.05
compared to saline/RA-exposed mice.
Fig. 1. Lung histology and morphometric analyses.
Lung sections from saline/RA, saline/O
2
, LPS/RA, and
LPS/O
2
-exposed mice were inflation fixed and hema
-
toxylin-and-eosin stained at 8 wk of age. A: represen-
tative images, stained with hematoxylin and eosin,
were taken at 100 magnification. Digital morpho-
metric analyses were performed on five images per
animal for number of alveoli (B) and septal thickness
(C). Data were analyzed using two-way ANOVA and
expressed as means SE; n 8 pups in each treat-
ment group, with no more than one animal from a
given litter per treatment group. *Significant differ-
ence, P 0.05 compared with saline/RA-exposed
mice; #Significant difference, P 0.05 compared with
LPS/RA-exposed mice. Significant difference P
0.05 compared to saline/O
2
. Scale bars 100 M.
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in mice exposed to LPS/O
2
(11,288 1,659 m
2
) than in
saline/RA controls. Septal thickness was 12% greater in the
saline/O
2
, 25% greater in the LPS/RA, and 39% greater in
the LPS/O
2
than in the saline/RA controls (Fig. 1C). In
summary, alveolar simplification and increased septal thick-
ness were evident in all treatment groups compared with
saline/RA controls; however, deficits in alveolarization and
increased septal thickness were most pronounced in mice
that received the combination of prenatal LPS and neonatal
hyperoxia.
Collagen deposition. Interstitial collagen deposition was
assessed in Mason’s trichrome-stained lung tissue sections at 8
wk of age. The blue color indicates collagen fibers in the lung
tissues. Saline/O
2
and LPS/RA-exposed mouse lungs showed
traces of blue staining within the lung tissue. However, sub-
stantial increases in blue staining are observed within the
tissues from LPS/O
2
-treated mice compared with all other
groups (Fig. 2A). Collagen deposition in blood vessels served
as a positive control in saline/RA mice. Collagen protein levels
were also quantified by Western blot analyses. No differences
in collagen I or collagen III levels were detected in saline/O
2
or
LPS/RA-exposed mice; however, our data indicate that colla-
gen I and III levels were 4 and 20 times greater, respectively,
in the lungs of LPS/O
2
-exposed mice compared with saline/RA
controls (Fig. 2B).
Collagen deposition was also quantified histologically. Ad-
ditional tissue sections were stained with Picrosirius red and
were observed under polarized light. Red (collagen I) or green
(collagen III) fluorescence was quantified using digital analysis
software. Significantly more red and green fluorescence was
detected in the LPS/O
2
(red 6.0 0.5; green 34.5 2.0 m
2
)
mice than any other group. There were no differences in
fluorescence intensities among the saline/O
2
(red 2.8 0.3;
green 21.5 1.4 m
2
), LPS/RA (red 2.1 0.2; green 16.7
0.9 m
2
), or the saline/RA (red 3.6 0.5; green 23.8 2.3
m
2
) groups. Both red and green fluorescence was signifi
-
cantly lower in LPS/RA-exposed mice compared with sa-
line/RA controls (Fig. 2C).
Fig. 2. Collagen deposition. Inflation-fixed lung sections from 8-wk-old mice were stained with Mason’s trichrome stain. A: representative images of saline/RA,
saline/O
2
, LPS/RA, and LPS/O
2
-exposed mice were taken at 100 magnification (scale bars represent 50 M). Western blots were performed on lung tissue
homogenates from 8-wk-old mice (n 5). B: representative blots and quantification of collagen I and collagen III proteins are as indicated. C: inflation-fixed
lung sections were stained with Picrosirius red and visualized under polarized light. Representative images were taken at 100 magnification. D: additional tissue
sections were stained Mac-3 antibody. Data were analyzed using two-way ANOVA and expressed as means SE; n 5–7 pups in each treatment group, with
no more than one animal from a given litter per treatment group; P 0.05 compared with saline/RA (*), saline/O
2
(†), and LPS/RA (#) -exposed mice.
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To assess the persistence of elevated pulmonary macrophage
numbers, lung tissue sections from mice at 8 wk of age were
stained with antimacrophage antibodies (Fig. 2D), and the
number of immunoreactive cells was counted. Saline/RA-
exposed mice showed only few [1.8 0.3 cells/high-power
field (HPF)], interstitial Mac-3-positive cells. While there was
no significant increase in the number of Mac-3-positive cells in
saline/O
2
(2.7 0.4 cells/HPF) or LPS/RA (4.2 0.5 cells/
HPF)-exposed mice, some positive stained cells were located
in the intra-alveolar space. However, LPS/O
2
-exposed mice
showed a significant increase (8.0 1.0 cells/HPF) in the
number of predominantly intra-alveolar Mac-3-positive stained
cells compared with all other groups.
Pulmonary function tests. Physiological consequences of
prenatal LPS and newborn hyperoxic exposure on adult pul-
monary functions were evaluated at 8 wk of age. PV-loops
were modestly upward shifted in LPS/RA-exposed mice com-
pared with saline/RA controls; however, this shift was even
more pronounced in LPS/O
2
mice (Fig. 3A).
Static compliance
and inspiratory capacity from zero pressure were significantly
lower in the LPS/O
2
mice than in all other groups (Fig. 3, B and
C). Tissue resistance was greater in LPS/O
2
than in saline/RA-
exposed mice (Fig. 3D).
MicroCt scans. Thoracic microcomputed tomography
(microCT) scans were performed on 8-wk-old mice and read
by a clinical radiologist blinded to group assignment. Com-
pared with saline/RA controls, the lungs of saline/O
2
, LPS/RA,
and LPS/O
2
mice were hyperexpanded, and the lung paren
-
chyma exhibited patchy centrilobular areas of alveolar simpli-
fication. However, hyperexpansion was most exaggerated, al-
veolar simplification was most widespread, and interstitial/septal
lung markings were coarsest in the LPS/O
2
group compared with
either saline/O
2
or LPS/RA-exposed mice (Fig. 4A).
Lung den-
sities were quantified and are displayed as Hounsfield unit
(HU) histograms (Fig. 4B). Compared with the saline/RA con-
trol histogram, the saline/O
2
histogram was shifted left, indicating
a less dense lung, while the LPS/O
2
histogram was shifted to the
right, indicating denser, fibrotic lung tissue. Three-dimensional
microCT reconstructions revealed multiple dense spots that were
only present in the lungs of LPS/O
2
-exposed mice (see Supple
-
mental Movies, LPS CO
2
vs. all others).
Fetal inflammatory response. mRNA levels of inflammatory
genes were assessed in fetal lung tissues to determine the
effects of maternal inflammation on the fetus. Fetal tissues
were collected on E18, which was 48 h after intraperitoneal
saline or LPS administration to the pregnant dam and the time
that most of the inflammatory responses measured were highest
in the fetus prior to birth (data not shown). TNF- and KC
mRNA levels were decreased (Figs. 5, A and D) and IL-1
mRNA was increased (Fig. 5B) in fetal lung tissues harvested
from LPS-injected dams compared with saline injected dams.
IL-6 and MCP-1 mRNA levels were not different between
groups (Fig. 5, C and E). Interestingly, TGF- and collagen I
mRNA levels were increased (Fig. 5, F and G) in fetal lungs
collected from LPS injected dams; however, collagen III
mRNA levels were not different (Fig. 5H).
Fig. 3. Pulmonary function analyzes. Using the
PVr-V perturbation from the FlexiVent (SCIREQ),
pulmonary function tests were generated in trache-
otomized mice at 8 wk of age. A: averaged pressure-
volume loops from saline/RA (black), saline/O
2
(blue), LPS/RA (green), and LPS/O
2
(red) exposed
mice. Analyzes of static compliance (Cst) (B) and
inspiratory capacity (C) from zero pressure. D: tis-
sue resistance was assessed using the Primewave-8
perturbation. A coefficient of determination of 0.98
was used as the lower limit for measurements. Three
correct maneuvers per mouse were averaged. Data
were analyzed using two-way ANOVA and ex-
pressed as means SE; n 6 litters; 12 pups in
each treatment group, with one male and one female
from each litter; P 0.05 compared with saline/RA
(*), saline/O
2
(†), and LPS/RA (#) -exposed mice.
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Neonatal inflammatory response during hyperoxia exposure.
Inflammatory responses to hyperoxia were evaluated in neo-
natal lung tissues from mice born to saline or LPS-injected
dams that were subsequently exposed to room air (RA) or 85%
O
2
. Tissues were harvested at 7 and 14 days, and mRNA and
protein expressions were analyzed. Data were analyzed and
compared with saline/RA controls at each corresponding time
point. TNF- mRNA levels were greater in LPS/O
2
-exposed
mice at 7 days and 14 days (Fig. 6A). IL-1 mRNA levels were
significantly greater in LPS/RA and LPS/O
2
-exposed mice at 7
days (Fig. 6A). IL-6 mRNA levels were greater in saline/O
2
- and
LPS/O
2
-exposed mice at 14 days (Fig. 6A). KC mRNA levels
Fig. 4. MicroCT imaging. MicroCT images
were assessed at 8 wk of age. A: representa-
tive high-resolution microCT images from
saline/RA, saline/O
2
, LPS/RA, and LPS/O
2
-
exposed mice. B: mean frequency histogram
of voxels having a particular Hounsfield unit
(HU). Five nonoverlapping areas were aver-
aged per mouse (n 3 pups in each treat-
ment group, with no more than one animal
from a given litter per treatment group).
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were greater at 14 days in saline/O
2
-exposed mice and at 7 days
and 14 days in LPS/O
2
-exposed mice (Fig. 6A). MCP-1 mRNA
levels were higher in LPS/O
2
-exposed mice at 14 days (Fig. 6A).
Expressions of inflammatory proteins were analyzed in lung
tissue homogenates using ELISA or Western blot. TNF-
protein levels were increased only in LPS/O
2
-exposed mice at
14 days compared with saline/RA controls (Fig. 6B). IL-6
protein levels were increased in LPS/O
2
-exposed mice at 14
days (Fig. 6B). KC protein levels were increased at 7 days and
14 days in both saline/O
2
and LPS/O
2
groups compared with
saline/RA controls (Fig. 6B). MCP-1 protein levels were higher
in LPS/O
2
-exposed mice at 14 days compared with saline/RA
controls (Fig. 6B).
Neonatal markers of fibrosis. mRNA levels of fibrotic mark-
ers were also assessed. TGF-1 levels were higher in all
treatment groups at 7 days and correlated with increases in
IL-1; however, increased levels were sustained only in LPS/
O
2
-exposed mice at 14 days (Fig. 7A).
Collagen I levels were
Fig. 5. Cytokine, chemokine, and collagen
mRNA expression in fetal lungs. Pregnant
mice were saline or LPS injected intraperi-
toneally on embryonic day 16 (E16). Fetal
lungs were harvested on E18, and pulmonary
mRNA expression of TNF- (A), IL-1 (B),
IL-6 (C), KC (D), MCP-1 (E), TGF- (F),
collagen I (G) and collagen III (H) was
analyzed. Expression was normalized to
-actin. Data were analyzed using t-test and
expressed as means SE; n 4 dams/
group, fetal lung tissues were pooled; *P
0.05 compared with saline/RA at the corre-
sponding time point.
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higher at 14 days in LPS/O
2
-exposed mice (Fig. 7A). Collagen
III levels were higher in all treatment groups at 7 days, but
increased levels were sustained only in LPS/O
2
exposed mice
at 14 days (Fig. 7A). As an indicator of TGF- activation, we
assessed protein levels of phospho-Smad 2 (p-Smad 2) by
Western blot analysis. p-Smad 2 was not different at 7 days
(Fig. 7B); however, levels were significantly increased at 14
days in saline/O
2
and greater in LPS/O
2
-exposed mice com
-
pared with saline/RA controls (Fig. 7B).
Levels of miR-29b were assessed in fetal lung tissues at
embryonic day 18 (E18) and at 14 days, the end point of the
hyperoxia exposure. miR-29b levels were significantly decreased
in fetal lung tissue harvested from LPS-injected dams compared
with tissues from saline-injected dams (Fig. 7C). At 14 days,
Fig. 6. Neonatal inflammatory response dur-
ing hyperoxia exposure. Pups born to E16
saline or LPS-injected dams were RA or
85% O
2
exposed. Mice were euthanized at 7
and 14 days, pulmonary mRNA and protein
isolated. A: expression of TNF-, IL-1,
IL-6, KC, and MCP-1 was analyzed by
quantitative real-time PCR and normalized
to -actin. B: protein expression of TNF-,
IL-6, KC, and MCP-1 was measured with
ELISA. Data were analyzed using two-way
ANOVA and Bonferroni post hoc and ex-
pressed as means SE; n 5 pups in each
treatment group, with no more than one
animal from a given litter per treatment
group; *P 0.05 compared with saline/RA
at the corresponding time point.
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miR-29b expression was lower only in the LPS/O
2
-exposed mice,
while miR-29b expression was greater in saline/O
2
and LPS/RA
groups compared with saline/RA controls (Fig. 7C).
DISCUSSION
The findings of the present study indicate that mouse lungs are
particularly vulnerable to perinatal inflammation. Using our pre-
viously established mouse model (53), we have identified path-
ways modulated by TGF- and miR29b expression. Additionally,
we observed deficits in alveolarization, diffuse interstitial fibrosis,
and impaired pulmonary function in exposed male and female
offspring as adults. Our data indicate that, in mice, activation of
proinflammatory and profibrotic pathways during the fetal and
neonatal period contributes to the development of an adult phe-
notype characteristic of pulmonary fibrosis in humans.
There is increasing recognition that hostile perinatal envi-
ronments influence fetal organogenesis and contribute to adult
diseases (47, 48, 53). Specifically, maternal infection and
Fig. 7. Neonatal fibrotic response during hy-
peroxia. Pups born to E16 saline or LPS-
injected dams were RA or 85% O
2
exposed.
Mice were euthanized at 7 and 14 days, and
pulmonary mRNA and protein were isolated.
A: expression of TGF-, collagen I and col-
lagen III was analyzed by quantitative real-
time PCR. B: protein expression of p-Smad2
and total Smad2/3 were assessed by Western
blot. C: pulmonary miRs were isolated, and
expression of miR29b was analyzed on em-
bryonic day 18 or at day 14. Expression was
normalized to Rnu6. Data were analyzed
using unpaired t-test or two-way ANOVA
and Bonferroni post hoc and expressed as
means SE; n 5 pups in each treatment
group, with no more than one animal from a
given litter per treatment group. *P 0.05
compared with saline/RA at the correspond-
ing time point.
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Page 9
inflammation are common during pregnancy and has adverse
repercussions on the developing fetus (25). The consequences
of intrauterine infections and inflammation have been exten-
sively studied in the context of early lung development (28,
30). However, recent evidence indicates that subtle systemic
maternal inflammatory responses likely influence both the
developing fetus and timing of parturition (5, 23, 44, 55). In
rodent models, low-dose systemic LPS administration induces
inflammatory responses in the pregnant dam (54). Maternally
derived inflammatory mediators rapidly cross the placenta and
are transmitted to the fetus, while only traces of maternally
administered LPS are detectable in the fetus (34, 46). Recent
advances in medical therapies have led to steadily increasing
survival rates in prematurely born infants that comprise a
unique population of children and young adults, many of which
have been exposed to both inflammation in utero and proin-
flammatory medical interventions after birth.
At 8 wk of age, mice exposed to LPS-induced maternal
inflammation and postnatal hyperoxia are slightly smaller in
total body weight but have relatively heavier lungs (Table 1),
despite dramatic decreases in alveolarization (Fig. 1). Deficits
in alveolarization are commonly observed with neonatal hy-
peroxic exposure in a dose-dependent manner (60) and are
evident in Fig. 1. Interestingly, maternal LPS exposure also
decreases alveolarization, and the decreases are even more
profound in the animals exposed to both insults. Given that a
large number of preterm infants are born to mothers with
inflammation (from a variety of sources), the additive effects of
maternal inflammation and neonatal hyperoxia could have
profound effects of the pulmonary function of the offspring in
later life.
The greater lung weights are most likely due to increased
collagen deposition detected in histological sections and in
lung homogenates (Fig. 2). While the position and temporal
expression of collagen are essential for normal lung growth;
just as important is the remodeling of the collagen network as
the lung grows and becomes more complex. Additionally,
macrophages can contribute to tissue remodeling and collagen
deposition. The persistent presence of increased numbers of
macrophages at 8 wk of age in the LPS/O
2
-treated mice
supports their role in the phenotype we have observed (Fig. 2).
Altered expression, cross-linking, or changes in the relative
amounts of collagen I and III are all associated with fibrotic
lung diseases and may be linked to dysregulation of cross-
linking enzymes (35, 47, 49). In the developing human lung,
collagen I and collagen III are weakly to moderately expressed
in the alveoli, arteries, veins, and adventitia (31). Redistribu-
tion of collagen protein precursors in the alveolar walls below
the alveolar epithelium or changes in expression of elastin/
collagen cross-linking enzymes are reported in infants diag-
nosed with BPD and in patients with idiopathic pulmonary
fibrosis (31, 41). Our Mason’s trichrome staining in tissues
(Fig. 2A) and the quantification of Picrosirius red staining (Fig.
2B) as well as protein levels indicate expression of collagen I
and III in the alveolar walls is much like those observed in
infants with BPD and in animal models of pulmonary fibrosis
(31, 41, 59). Consequently, we speculated that the increases in
collagen observed in our model are more likely a deficiency in
matrix remodeling rather than a dysregulation in deposition.
Pulmonary function tests in the present studies revealed
stiffer lung tissues in LPS/O
2
-exposed, mice as indicated by
reduced static compliance, reduced inspiratory capacity from
zero pressure, and increased tissue resistance (Fig. 3). A
similar reduction in static compliance was reported in a mouse
model of bleomycin-induced pulmonary fibrosis (13, 42). The
fact that the LPS/O
2
-exposed mice show alterations in pulmonary
mechanics similar to animals in an acute model of pulmonary
fibrosis highlights the significant physiological consequences of
perinatal exposures on lung development.
Lung structure was evaluated radiologically in adult mice
using microCT imaging. While our data revealed alveolar
simplification in all treatment groups, this was most severe in
LPS/O
2
-exposed mice, which is also indicated by increased
alveolar areas in histological sections. Consistent with previous
reports, saline/RA and LPS/RA-exposed mice had lung densi-
ties in the range of 450 to 550 HU (24, 40). Interestingly,
the saline/O
2
-exposed group had lower lung densities (leftward
shift) that are consistent with our histological findings of fewer
and larger alveoli and are similar to that seen clinically in adult
patients with emphysema (39). Conversely, LPS/O
2
-exposed
mice had lung densities that were greater than controls (right-
ward shift), which is indicative of more dense lung tissues.
Increases in lung densities measured by microCT have been
reported in animal models of pulmonary fibrosis (24, 40), as
well as in human patients (50). Greater densities in the present
studies are supported histologically and biochemically by the
observed increases in collagen deposition in the LPS/O
2
group
(Fig. 2).
Inflammation can disrupt developmental pathways, and our
data indicate alterations in mRNA expression levels of proin-
flammatory and profibrotic genes in fetal lung tissues obtained
from LPS-injected dams. Surprisingly, TNF- and KC mRNA
levels were lower in fetal lung tissues harvested from LPS-
injected dams (Fig. 6). We speculate that E18, which was 48 h
postinjection, was too late to detect transient increases in the
mRNAs expression of these molecules. However, IL-1 was
substantially elevated at E18 and correlated with increased
levels of TGF- and collagen I in fetal lung tissues from
LPS-injected dams. Our current findings indicate that maternal
LPS injection induced fetal inflammatory response, making the
organism more vulnerable to subsequently occurring insults (6).
mRNA expression and protein levels of mediators that
regulate leukocyte infiltration were increased during neonatal
hyperoxic exposure. KC, TNF-, IL-6, and MCP-1 mRNA
expression and protein levels were increased in saline/O
2
and
LPS/O
2
-exposed mice throughout the hyperoxic exposure pe
-
riod compared with RA controls (Fig. 6). While MCP-1, IL-6,
and TNF- protein levels were lower in saline/O
2
mice by 14
days, they remained elevated in LPS/O
2
mice. Increases in IL-6
mRNA expression and protein levels have been previously
reported in the offspring of rats exposed to intra-amniotic LPS
and neonatal hyperoxia (33) and in human infants born to
mothers with diagnosed chorioamnionitis (44). However, our
model differs from these studies in that elevations in IL-6
levels are more closely linked to hyperoxia exposure than to
the combination of LPS/O
2
(Fig. 6).
Collagen expression and fibrosis are regulated by TGF-
and/or Smad-related signaling (21), potentially through mod-
ulation of miR-29b expression (12). The TGF- pathway has
been implicated in development of BPD, as well as other
fibrotic lung diseases (21). In addition to its role in matrix
remodeling and fibrosis, TGF- plays a key role in alveolar
R288 FETAL ORIGINS OF ADULT PULMONARY DISEASE
AJP-Regul Integr Comp Physiol doi:10.1152/ajpregu.00029.2012 www.ajpregu.org
Page 10
development and maintenance of alveolar structure (1). Devel-
opmental overexpression of TGF- results in impaired alveo-
larization and Smad-dependent interstitial fibrosis in monkeys
(51) and rats (20, 21). Increases in IL-1, TGF-, and collagen
I mRNA levels were persistent after birth in hyperoxia-exposed
mice. There were additional increases in collagen III in hyper-
oxia-exposed mice, particularly at 7 days. By day 14, IL-1
levels were similar to controls but TGF- and collagen I and III
mRNA expression remained elevated solely in the LPS/O
2
-
exposed mice (Figs. 6 and 7). Additionally, elevated levels in
TGF- and collagen I and III mRNA were associated with
increases in p-Smad2 protein levels in only the LPS/O
2
ex
-
posed at day 14.
Previous investigations have linked macrophage-induced
increased IL-1 levels with altered TGF- and Smad expres-
sions, and subsequent collagen deposition and fibrosis, sug-
gesting that this phenotype more closely resembles impaired
tissue repair rather than acute injury (6, 37). One explanation of
our current findings could be that the LPS exposure in utero
initiates a lung injury that is either unable to resolve or
interrupted during the healing process by the inflammatory
responses to hyperoxia exposure. Regardless, the sustained
increases in MCP-1, IL-6, TNF-, IL-1, TGF-, and collagen
I and III, distinguish the LPS/O
2
from the other treatment
groups and are, in part, responsible for the physiological
deficits in lung structure and function observed at 8 wk.
Our data provide the first evidence that TGF- mRNA levels
in fetal lungs are increased by LPS-induced maternal inflam-
mation and that subsequent hyperoxia exposure caused the
induction to persist. TGF- regulation of downstream profi-
brotic genes has been linked to suppression of miR-29b ex-
pression (12). Our data indicate that miR-29b is decreased at
E18 in fetal lung tissues from LPS-treated dams (Fig. 7) and is
inversely correlated with TGF- and collagen I and III expres-
sions. On day 14, miR-29b expression was further suppressed
in LPS/O
2
mice; however, miR-29b expression was elevated in
saline/O
2
compared with saline/RA mice probably as a com
-
pensatory response. miR-29b reduces expression of proteins
that are regulated by TGF- stimulation, such as collagen I and
III (12). We hypothesize that elevated mRNA levels of colla-
gen I and III in LPS/O
2
on E18 and 14 days are due to
suppression of miR-29b levels. Whether directly or indirectly
through miR-29b, our data suggest that TGF- expression is
fundamentally involved in the fibrotic phenotype observed in
the LPS/O
2
-exposed mice.
In summary, the present study demonstrates that systemic
maternal LPS administration induces a profibrotic response in
fetal lung tissue that begins prior to birth and that results in an
ongoing activation of profibrotic pathways when combined
with neonatal hyperoxic exposure. These dual inflammatory
exposures lead to the development of diffuse interstitial fibro-
sis, impaired alveolarization, and compromised pulmonary
mechanics in both adult male and female offspring with no
differences between sexes. Our data suggest that these changes
are mediated through reduction in miR-29b expression, result-
ing in activation of profibrotic pathways, including, but not
exclusive to, TGF--mediated signaling. The finding that peri-
natal exposures result in persistently impaired pulmonary phys-
iology in adulthood provides a novel model to investigate the
influence of the perinatal environment on the development of
adult pulmonary diseases.
ACKNOWLEDGMENTS
The authors would like to acknowledge and thank Dr. Loren Wold for his
editorial assistance and Dr. Frederick Long for radiologic interpretations of the
microCT scans.
GRANTS
This work was supported by the Deutsche Forschungsgemeinschaft (VE
614/1-1) and the National Institutes of Health (RDB F31HL097619, TET
1K08HL093365-01A2 and LKR R01AT006880).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: M.V., S.E.W., T.E.T., and L.K.R. conception and
design of research; M.V., R.D.B.J., K.M.H., and B.E. performed experiments;
M.V., R.D.B.J., K.M.H., and L.K.R. analyzed data; M.V., R.D.B.J., T.E.T.,
and L.K.R. interpreted results of experiments; M.V. and L.K.R. prepared
figures; M.V. and L.K.R. drafted manuscript; M.V., R.D.B.J., B.E., T.E.T., and
L.K.R. edited and revised manuscript; M.V., R.D.B.J., S.E.W., B.E., T.E.T.,
and L.K.R. approved final version of manuscript.
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R290 FETAL ORIGINS OF ADULT PULMONARY DISEASE
AJP-Regul Integr Comp Physiol doi:10.1152/ajpregu.00029.2012 www.ajpregu.org
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  • [Show abstract] [Hide abstract] ABSTRACT: Supraphysiological O2 concentrations, mechanical ventilation, and inflammation significantly contribute to the development of bronchopulmonary dysplasia (BPD). Exposure of newborn mice to hyperoxia causes inflammation and impaired alveolarization similar to that seen in infants with BPD. Previously, we demonstrated that pulmonary cyclooxygenase-2 (COX-2) protein expression is increased in hyperoxia-exposed newborn mice. The present studies were designed to define the role of COX-2 in newborn hyperoxic lung injury. We tested the hypothesis that attenuation of COX-2 activity would reduce hyperoxia-induced inflammation and improve alveolarization. Newborn C3H/HeN mice were injected daily with vehicle, aspirin (non-selective COX-2 inhibitor), or celecoxib (selective COX-2 inhibitor) for the first 7 days of life. Additional studies utilized wild type (C57Bl/6, COX-2(+/+)), heterozygous (COX-2(+/-)), and homozygous (COX-2(-/-)) transgenic mice. Mice were exposed to room air (21% O2) or hyperoxia (85% O2) for 14 days. Aspirin-injected and COX-2(-/-) pups had reduced levels of monocyte chemoattractant protein (MCP-1) in bronchoalveolar lavage fluid (BAL). Both aspirin and celecoxib treatment reduced macrophage numbers in the alveolar walls and airspaces. Aspirin and celecoxib treatment attenuated hyperoxia-induced COX activity, including altered levels of prostaglandin (PG)D2 metabolites. Decreased COX activity, however, did not prevent hyperoxia-induced lung developmental deficits. Our data suggests that increased COX-2 activity may contribute to pro-inflammatory responses, including macrophage chemotaxis, during exposure to hyperoxia. Modulation of COX-2 activity may be a useful therapeutic target to limit hyperoxia-induced inflammation in preterm infants at risk of developing BPD.
    No preview · Article · Apr 2013 · Free Radical Biology and Medicine
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  • [Show abstract] [Hide abstract] ABSTRACT: Wheezing and asthma are significant clinical problems for infants and young children, particularly following premature birth. Recurrent wheezing in infants can progress to persistent asthma. As in adults, altered airway structure (remodeling) and function (increased bronchoconstriction) are also important in neonatal and pediatric airway diseases. Accumulating evidence suggests that airway disease in children is influenced by perinatal factors including perturbations in normal fetal lung development, postnatal interventions in the intensive care unit (ICU) and environmental and other insults in the neonatal period. Here, in addition to genetics, maternal health, environmental processes, innate immunity and impaired lung development/function can all influence pathogenesis of airway disease in children. We summarize current understanding of how prenatal and postnatal factors can contribute to development of airway diseases in neonates and children. Understanding these mechanisms will help identify and develop novel therapies for childhood airway diseases.
    No preview · Article · Oct 2013 · Expert Review of Respiratory Medicine
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  • [Show abstract] [Hide abstract] ABSTRACT: In contrast to early lung development, a process exemplified by the branching of the developing airways, the later development of the immature lung remains very poorly understood. A key event in late lung development is secondary septation, in which secondary septa arise from primary septa, creating a greater number of alveoli of a smaller size, which dramatically expands the surface area over which gas exchange can take place. Secondary septation, together with architectural changes to the vascular structure of the lung which minimize the distance between the inspired air and the blood, are the objectives of late lung development. The process of late lung development is disturbed in bronchopulmonary dysplasia (BPD), a disease of prematurely-born infants in which the structural development of the alveoli is blunted as a consequence of inflammation, volutrauma, and oxygen toxicity. This review aims to highlight notable recent developments in our understanding of late lung development and the pathogenesis of BPD.
    Preview · Article · Nov 2013 · AJP Lung Cellular and Molecular Physiology
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