Antenatal Inflammation Reduces Expression of
Caveolin-1 and Influences Multiple Signaling
Pathways in Preterm Fetal Lungs
Steffen Kunzmann1, Jennifer J. P. Collins2, Yang Yang3, Stefan Uhlig3, Suhar G. Kallapur4,5,
Christian P. Speer1, Alan H. Jobe4,5, and Boris W. Kramer2
1University Children’s Hospital, University of Wu ¨rzburg, Wu ¨rzburg, Germany;2School of Mental Health and Neuroscience, and Department of
Pediatrics, School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands;3Institute of Pharmacology and
Toxicology, Medical Faculty, Rheinisch-Westfa ¨lische Technische Hochschule Aachen University, Aachen, Germany;4Division of Pulmonary Biology,
Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio; and5School of Women’s and Infants’ Health, University of Western Australia, Perth,
Western Australia, Australia
Bronchopulmonary dysplasia (BPD), associated with chorioamnio-
nitis, results from the simultaneous effects of disrupted lung de-
velopment,lung injury,andrepair superimposedonthedeveloping
lung. Caveolins (Cavs) are implicated as major modulators of lung
injury and remodeling by multiple signaling pathways, although
Cavs have been minimally studied in the injured developing lung.
We hypothesized that chorioamnionitis-associated antenatal lung
inflammation would decrease the expression of Cav-1 in preterm
activation of acid-sphingomyelinase (a-SMase) with the generation
of ceramide, along with changes in the expression of heme oxygen-
ase–1 (HO-1) as indicators of possible Cav-1–mediated effects. Fetal
sheep were exposed to 10 mg of intra-amniotic endotoxin or saline
for 2, 7, or 2 1 7 days before preterm delivery at 124 days of gesta-
tion. The expression of Cav-1 and HO-1 and the phosphorylation of
Smad and Stat were evaluated by real-time PCR, Western blotting,
centrations of ceramide were measured. Intra-amniotic endotoxin
decreased Cav-1 mRNA and protein expression in the lungs, with
a maximum reduction of Cav-1 mRNA to 50% 6 7% of the control
control value (P , 0.05). Decreased concentrations of Cav-1 were
associated with the elevated phosphorylation of Smad2/3, Stat3,
and Stat1, but not of Smad1/5. The expression of HO-1, a-SMase
the expression of Cav-1 in the preterm fetal lung. The decreased ex-
pression of Cav-1 was associated with the activation of the Smad2/3,
sion of HO-1. The decreased concentrations of Cav-1 and changes in
other signaling pathways may contribute to BPD.
Keywords: bronchopulmonary dysplasia; TGF-b; a-SMase; ceramide;
Lung inflammation is a major contributor to the impaired devel-
pulmonary dysplasia (BPD) (1). However, the mechanisms that
link inflammation to alveolar and microvascular simplification
are unclear (2). For many very preterm infants, pulmonary in-
flammation begins in utero with chorioamnionitis. Chorioamnio-
and is an important risk factor for preterm delivery in approxi-
mately 60% of very preterm deliveries (3). Chorioamnionitis
increases proinflammatory cytokines in human amniotic fluid
and fetal cord blood, presumably by fetal responses to bacterial
important mediators that recruit activated inflammatory cells to
the fetal lung (2). Fetal sheep develop chorioamnionitis after
injections of LPS into the amniotic fluid, which initiates a se-
quence of lung injury (inflammation, apoptosis, and remodeling)
that results in both lung maturation and decreased alveolar sep-
tation with microvascular injury (5). These changes in the fetal
lung may initiate the progression to BPD (6).
Caveolin-1 (Cav-1) may be central in this pathophysiolo-
gical sequence as a component of caveolae, which are 50–
100-nm-wide omega-shaped plasma membrane invaginations
(7). Caveolae and caveolins are present at high concentrations
in the airway epithelium, smooth muscle, fibroblasts, inflamma-
tory cells, and pulmonary vasculature (8). Caveolae function in
protein trafficking, signal transduction, and sphingolipid biology
(9).Thedown-regulationofCav-1occurs indiverselung diseases
such as asthma, chronic obstructive pulmonary disease, and idio-
LPS-induced lung injury in animal models (10).
At the molecular level, Cav-1 is fundamental in organizing
(11, 12), Stat (13–15), and acid-sphingomyelinase (a-SMase)/
in airway inflammation and remodeling (18–22). Cav-1 also reg-
ulates inducible heme oxygenase–1 (HO-1) (23), which modu-
lates oxidative and inflammatory defenses in the lung (24).
Changes in Cav-1 or associated changes in these signaling path-
(Received in original form December 26, 2010 and in final form April 14, 2011)
This work was supported by Deutsche Forschungsgemeinschaft grant KU 1403/
2–1 (S.K.), by University of Wu ¨rzburg Interdisciplinary Center for Clinical Re-
search grants IZKF Z-08 and A-58 (S.K.), by National Heart, Lung, and Blood
Institute grant HL-65397, by the Dutch Scientific Research Organization, by
the School of Mental Health and Neuroscience and School for Oncology and
Developmental Biology, Maastricht University, and by a VENI grant from the
Netherlands Organisation for Scientific Research (B.W.K.).
S.K., J.J.P.C., Y.Y., S.U., S.G.K., A.H.J., and B.W.K. were responsible for the con-
ception and design of this study. Analyses and interpretation were performed by
S.K., J.J.P.C., Y.Y., S.U., S.G.K., A.H.J., and B.W.K. Drafting the manuscript for
important intellectual content was performed by S.K., B.W.K., A.H.J., and C.P.S.
Correspondence and requests for reprints should be addressed to Steffen
Kunzmann, M.D., University Children’s Hospital, Josef-Schneider Strasse 2,
97080 Wu ¨rzburg, Germany. E-mail: firstname.lastname@example.org
This article has an online supplement, which is accessible from this issue’s table of
contents at www.atsjournals.org
Am J Respir Cell Mol Biol
Originally Published in Press as DOI: 10.1165/rcmb.2010-0519OC on May 11, 2011
Internet address: www.atsjournals.org
Vol 45. pp 969–976, 2011
The study supports a role for Cav-1 in lung remodeling
induced by antenatal inflammation in BPD.
We previously reported increased TGF-b1 activity in fetal
lungs after antenatal exposure to inflammation (25). TGF-b1
was identified in lung fibroblasts and endothelial cells as a nega-
tive regulator for Cav-1 (26–28). We therefore hypothesized that
antenatal inflammation would decrease the expression and func-
tion of Cav-1 in preterm lungs, thereby affecting the Smad, Stat,
nionitis to cause inflammation in fetal lungs (29). A better un-
derstanding of the signal transduction pathways in fetal lung
inflammation may provide new therapeutic approaches to the
treatment of postnatal lung injury.
MATERIALS AND METHODS
All animal experiments were performed in Western Australia with the
approval of the Ethics Committees of the Department of Agriculture
of Western Australia and the Children’s Hospital Research Foundation
in Cincinnati, Ohio. Time-mated ewes with singletons were assigned to
groups of six or seven animals for ultrasound-guided intra-amniotic
injections of LPS (10 mg, Escherichia coli O55:B5; Sigma Chemicals,
St, Louis, MO) in 2 ml saline, 2, 7, or 2 1 7 days before delivery
(Figure 1). Control animals received a 2-ml intra-amniotic injection
of saline. No differences were evident among the control animals that
received saline injections at different time points before delivery.
Therefore, the control animals were combined into one group. All
animals were operatively delivered at the same gestational age of
124 days (term, 150 days). Lung tissue was used for multiple assess-
ments. Results in terms of lung inflammation and maturation were
previously reported for these animals (30).
Immunohistochemistry and Histologic Analysis
The immunostaining methods were previously described (31) (please
see the online supplement for details).
Measurements of a-SMase and Ceramide
The activity of a-SMase was determined with14C-labeled sphingomye-
lin (32). Powdered lung was mixed with a-SMase buffer (250 mM
Na-acetate, 1 mM EDTA, and 0.1% Triton X-100, pH 5.0) and
homogenized. Samples were centrifuged at 4˚C and 20,000 3 g for 20
minutes, and the protein content of the supernatant was determined.
Samples were incubated at 378C for 2 hours with14C-labeled sphingo-
myelin substrate. Samples were separated by chloroform/methanol ex-
traction, scintillation liquid was added, and radioactivity was measured.
Concentrations of ceramide in lung tissue and serum were determined
as described elsewhere (33). In brief, powdered lung tissue was mixed
in a methanol/chloroform water emulsion, sonicated, and centrifuged
at 48C and 4,000 3 g for 10 minutes to extract the lipids. Lipids were
separated from other membrane components by chloroform/methanol
extraction and dried with N2. Subsequently, lipids were dissolved in
chloroform/methanol (9:1) and spotted on high-performance thin-
layer chromatography plates (Silica Gel 60 Precoated Plates; Merck,
Darmstadt, Germany). Ceramide was resolved with dichloromethane/
methanol/acetate (100:2:5). Thin-layer chromatography plates were
dried at 1808C, cooled, and immersed in a solution of 10% cupric
sulfate and 8% phosphoric acid. After heating for 2 minutes at
1108C, lipid bands became visible and were measured with a Fujix-
1000 Bioimager (Raytest, Straubenhardt, Germany).
For the extraction of RNA, PCR and Western blotting conditions,
and statistical analyses, please see the online supplement.
Pulmonary Expression of Cav-1 Is Reduced by
The results of PCR, expressed as Cav-1 mRNA transcripts by
PCR normalized to ovine ribosomal protein S15, showed that
LPS reduced Cav-1 mRNA by 50% in the 2-day, 7-day, and
2 1 7–day LPS groups relative to the control group (P ,
0.05) (Figure 2A). To confirm that the decreased expression
of Cav-1 mRNA corresponded with reduced protein concentra-
tions, Cav-1 was quantified by Western blot (Figures 2B and
2C) and immunohistochemistry (Figures 2D–2F) analysis.
Cav-1 decreased similarly at 2 days and 7 days with these meas-
urements, and a second exposure to LPS exerted no further
effect on the expression of Cav-1 protein.
Smad2/3 but Not Smad1/5 Phosphorylation Increase with
Cytoplasmic staining for phosphorylated Smad2/3 was weak in
bronchial epithelial cells in control lungs (Figure 3A). In con-
trast, exposure to LPS resulted in intense, phosphorylated
Smad2/3 staining (Figure 3B). The phosphorylated Smad2/3
staining increased 2 days after exposure to LPS, and increased
further by approximately threefold, 7 days after exposure to
LPS (Figure 3C). The majority of cells exhibited nuclear stain-
ing (Figure 3B, inset and arrow), consistent with TGF-b1 signal-
ing and the nuclear translocation of phosphorylated Smad2/3.
Only weak staining was detected for phosphorylated Smad1/5
in LPS-treated and control animals (Figure 3D).
Phosphorylation of Stat3 and Stat1 by
Concentrations of phosphorylated Stat3 increased in the 2-day,
7-day, and 2 1 7–day LPS-exposed animals according to West-
ern blot analysis, compared with control animals (Figure 4A). A
semiquantitative analysis of immunoblots demonstrated that
phosphorylated Stat3 was induced 355% (P , 0.05) in the 2
1 7–day LPS-exposed group, compared with the control group
(Figure 4B). The phosphorylation of Stat3 was also detected
Stat3-specific antibodies (Figures 4C–4E). Staining for phos-
phorylated Stat3 was weak in control lungs (Figure 4C). In
contrast, exposure to LPS resulted in the intense staining of
Figure 1. Study design. Six to seven animals per group received ultra-
sound-guided intraamniotic injections with 10 mg LPS or NaCl 0.9%
(control), 2 days, 7 days, or 2 1 7 days before delivery at the same
gestational age of 124 days. d, days.
970AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 452011
phosphorylated Stat3 (Figure 4D). A semiquantitative analysis
of the immunohistochemistry demonstrated that phosphory-
lated Stat3 increased approximately fourfold (P , 0.05) in the
7-day and 2 1 7–day LPS-exposed group, compared with the
control group (Figure 4E).
analysis, a 4.8-fold increase (Figures 5C–5E) was also measured
after 2 days of exposure to LPS, relative to control values (P ,
exhibited nuclear staining, consistent with translocation to the
nucleus (Figures 4D and 5D, insets and arrows).
Activity of a-SMase and Concentrations of Ceramide Increase
with LPS-Induced Chorioamnionitis
In the LPS-exposed groups, both the activity of a-SMase and
concentrations of ceramide were elevated, compared with con-
trol values (Figure 6). The activity of a-SMase increased 18-fold
in the 7-day LPS-exposed group (Figure 6A; P , 0.05), and
concentrations of ceramide increased 38-fold in the 7-day
LPS-exposed group (Figure 6B; P , 0.05).
Induction of HO-1 Expression by
According to Western blot analysis, the ratio of HO-1 protein to
b-actin protein increased 4.5-fold in the 7-day LPS-exposed
group compared with the control group (P , 0.05) (Figures 7A
and 7B). HO-1 protein was increased in all three experimental
groups according to immunohistochemical analysis, with a maxi-
with the control group (P , 0.05) (Figures 7C–7E).
Chorioamnionitis caused LPS-induced lung inflammation and
airway remodeling (34, 35). This fetal inflammatory response
also down-regulated Cav-1, activated Stat, Smad, and a-SMase,
and up-regulated the expression of ceramide and HO-1 in the
fetal lung. These findings support the hypothesis that the de-
creased expression of pulmonary Cav-1 and effects on down-
stream signaling pathways may contribute to remodeling and
functional impairment in the developing lung.
Animal models of LPS-induced chorioamnionitis were exten-
sively used to evaluate the effects of inflammation on lungs and
other organs (36–38) and potential mediators (39–41). Intra-
amniotic LPS can reliably reproduce the fetal inflammatory
response in chorioamnionitis, but with the limitation that cho-
rioamnionitis is induced with only one proinflammatory Toll-
like receptor ligand and not with living bacterial infections,
which are often polymicrobial in chorioamnionitis (42).
lung, because in human pulmonary fibroblasts, TGF-b1 can
the treatment of cultured bovine aortic endothelial cells with
Figure 2. Intra-amniotic exposure to LPS decreases expression of
caveolin-1 (Cav-1). (A) Real-time PCR measurements of Cav-1 mRNA
expression in whole-lung homogenates. Mean fold change in lung
mRNA expression of Cav-1 normalized for ovine ribosomal protein
S15 by the DDCtmethod. (B) Western blot measurements of Cav-1
protein expression with an anti–Cav-1 antibody. The same membrane
was analyzed with anti–b-actin antibody. (C) Concentrations of Cav-1
and b-actin protein were semiquantified by densitometry. Optical den-
sity of Cav-1 protein band was corrected to b-actin, and results are
expressed as ratio (%) of endotoxin-exposed animals to control ani-
mals. (D–F) Immunohistochemical evaluation of Cav-1 expression in
preterm lung tissue. Sections are representative of a control animal
(D) and an animal exposed to LPS-induced chorioamnionitis for 2 days
(E), stained for Cav-1. Magnification, 350. (F) Immunostaining for
Cav-1 was graded on a scale from 0–3. Values are means 6 SE. *P ,
0.05, versus control group.
Kunzmann, Collins, Yang, et al.: Regulation of Cav-1 by Inflammation in Preterm Fetal Lungs 971
the down-regulation of Cav-1 mRNA expression by TGF-b1 in
A549 cells (an adenocarcinoma human alveolar epithelial cell
line), and in H441 cells (a human lung adenocarcinoma cell line
in the lungs of fetal lambs (25), and concentrations of TGF-b1
were increased in airway samples from preterm infants develop-
chorioamnionitis-induced inflammation may be mediated by in-
creased concentrations of TGF-b1 in the lung (25). Although the
Figure 3. Effect of intra-amniotic exposure to LPS on Smad signaling.
The phosphorylation of Smad2/3 (A–C) and Smad1/5 (D) was evalu-
ated in lung tissue by immunohistochemistry. Sections are representa-
tive of a control animal (A) and an animal exposed to LPS-induced
chorioamnionitis for 7 days (B), stained for phosphorylated Smad2/3.
Magnification, 350. Inset shows higher magnification, and arrow iden-
tifies nuclear staining for phosphorylated Smad2/3. (C) Immunohisto-
chemical semiquantification of phosphorylated Smad2/3 in lung
sections. (D) Immunohistochemical semiquantification of phosphory-
lated Smad1/5 in lung sections. Immunostaining for phosphorylated
(P) Smad2/3 or Smad1/5 was graded on a scale from 0–3. Values
represent means 6 SE. *P , 0.05, versus control group.
Figure 4. Effect of intra-amniotic exposure to LPS on Stat3 signaling.
(A) Western blot measurements of Stat3 phosphorylation with an anti-
phosphorylated Stat3 antibody. The same membrane was analyzed
with anti–b-actin antibody. Phosphorylated Stat3 and b-actin protein
concentrations were semiquantified by densitometry. (B) Optical den-
sity of phosphorylated Stat3 protein bands was corrected to b-actin,
and results are expressed as the ratio (%) of LPS-exposed animals to
control animals. (C–E) Evaluation of Stat3 phosphorylation in lung tis-
sue by immunohistochemistry. Sections are representative of a control
animal (C) and an animal exposed to LPS-induced chorioamnionitis for
7 days (D), stained for phosphorylated Stat3. Magnification, 350. Inset
shows higher magnification, and arrow identities nuclear staining for
phosphorylated Stat3. (E) Immunohistochemical semiquantification of
phosphorylated Stat3 in lung sections. Values represent means 6 SE.
*P , 0.05, versus control group.
972 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 45 2011
down-regulation of Cav-1 was described previously in other lung
diseases (10), the reduced expression of Cav-1 in preterm lung
disease constitutes a new observation.
In this translational model of fetal sheep, we cannot directly
demonstrate that TGF-b1 was regulating Cav-1. Cav-1 also con-
tributes to the regulation of TGF-b signaling by its participation
in the internalization of TbR (TGF-b receptor) (45). TbRs can
be internalized by two different mechanisms, either by Cav-1
associated lipid rafts, or by early endosome antigen–1 nonlipid
raft pathways. Whereas nonlipid raft–associated internalization
increases TGF-b signaling, caveolin-associated internalization
increases the degradation of TbR and decreases TGF-b signal-
ing (46). The absence of one compartment or an imbalance in
the densities of the two compartments may affect the level of
TGF-b pathway activity, given the same amount of ligand bind-
ing. Because this process occurs at the level of internalization of
the TbR immediately after ligand engagement, it likely repre-
sents an important mechanism for regulating TGF-b signaling.
In human fetal pulmonary fibroblasts, experiments on both the
gain and loss of function identified the regulatory role of Cav-1
in this process (28). The down-regulation of Cav-1 by siRNA
transfection increased the phosphorylation of Smad-2 and of
Smad2/3 nuclear translocation, whereas the overexpression of
Cav-1 suppressed the phosphorylation of Smad2 and nuclear
translocation (28). In addition, enhanced TGF-b signaling
was measured in Cav-1–deficient mice (47) and in ovalbumin
allergen–challenged Cav-1–deficient mice in a model of asthma
(48). Furthermore, Razani and colleagues described an interac-
tion between Cav-1 and the Type I TGF-b receptor, in which
Cav-1 suppressed the TGF-b1–mediated phosphorylation of
Figure 6. Effects of intra-amniotic exposure to LPS on acid-sphingo-
myelinase (a-SMase) activity and ceramide. (A) Measurements of a-
SMase activity in lung tissue by a modified micellar in vitro assay. (B)
Measurements of ceramide concentrations by two-dimensional char-
ring densitometry. Values represent means 6 SE. *P , 0.05, versus
Figure 5. Effect of intra-amniotic exposure to LPS on Stat1 signaling.
(A) Western blot measurements of Stat1 phosphorylation with an anti-
phosphorylated Stat1 antibody. The same membrane was analyzed
with anti–b-actin antibody. Concentrations of phosphorylated Stat1
and b-actin protein were semiquantified by densitometry. (B) Optical
density of phosphorylated Stat1 protein band was corrected for b-actin,
and results are expressed as the ratio (%) of LPS-exposed animals to
control animals. Values represent means 6 SE. *P , 0.05, versus control
group. (C–E) Evaluation of Stat1 phosphorylation in lung tissue by
immunohistochemistry. Representative sections from a control animal
(C) and an animal exposed to LPS-induced chorioamnionitis for 2 days
(D), stained for phosphorylated Stat1. Magnification, 350. Inset shows
higher magnification, and arrow indicates nuclear staining for phos-
phorylated Stat1. (E) Immunohistochemical semiquantification of
phosphorylated Stat1 in lung sections. Values represent means 6 SE.
*P , 0.05, versus control group.
Kunzmann, Collins, Yang, et al.: Regulation of Cav-1 by Inflammation in Preterm Fetal Lungs973
Smad2 (11). We analyzed the phosphorylation of Smad2/3 and
Smad1/5 in preterm lungs. Smads are downstream effectors of
TGF-b, and provide an indication of the extent to which TGF-
b signaling is activated. In concordance with other studies, the
decreased expression of Cav-1 in our model was associated with
a substantial increase in phosphorylated Smad2/3 in the lungs of
animals exposed to LPS. The increase of Smad2/3 phosphoryla-
tion is in agreement with an earlier similar study with different
intervals of exposure to LPS (25).
a latent form in the cytoplasm, and upon receptor activation by
tyrosine kinase 2 (Tyk2) (49). An activated Stat pathway is com-
monly observed in acute lung injury (e.g., in endotoxin-induced
mice (14). In addition, the Jak/Stat signaling cascade is hyper-
activated in the mammary glands of Cav-12/2mice (50). Thus,
the down-regulation of Cav-1 expression should hyperactivate
the phosphorylation of Stat3. In our model of LPS-induced lung
injury, the reduced expression of Cav-1 was associated with in-
creased phosphorylation of Stat-3 and Stat-1.
The a-SMase/ceramide pathway is critical in a variety of lung
diseases (21), including acute neonatal inflammatory lung injury
(22). Sphingolipids are structure-bearing elements of biological
membranes that regulate key physiological processes such as ap-
optosis, innate and acquired immunity, vascular permeability,
and smooth muscle tone (21). Ceramide is a structure-bearing
lipid and probably also a second messenger that is generated by
Consistent with interactions between a-SMase and Cav-1 (16, 17,
a-SMase activity and the production of ceramide, as observed in
many models of acute lung injury (21). The decreased expression
of a-SMase/ceramide afterrepetitive injections of endotoxin into
the amniotic fluid may be part of the endotoxin tolerance that we
previously reported in this model (30).
injury, because of the known association between the decreased
expression of Cav-1 and increased expression of HO-1 in the
lung (23, 52). Like the Smad and Stat signaling pathways, HO-1
can influence pulmonary remodeling (24). HO-1 is a mediator of
tissue protection against a wide variety of injurious insults (24).
The increased expression of HO-1 was described in premature
infants with respiratory distress syndrome (53). In addition,
Maroti and colleagues suggested that HO-1 plays a role in the
days after birth (54). A direct link between Cav-1 and HO-1 was
characterized byJin andcolleagues (23) when they examined the
underlying mechanisms by which Cav-12/2mice manifested pro-
longed survival and reduced lung injury after hyperoxia. The ap-
parent resistance to hyperoxia in Cav-12/2pulmonary cells and
tissues resulted from the increased expression of stress protein
HO-1 (23). Furthermore, Kim and colleagues demonstrated that
the activity of HO-1 dramatically increased in endothelial cells
expressing Cav-1 antisense transcripts, suggesting a negative reg-
ulatory role for Cav-1 (52).
In parallel with these observations, the connections between
Cav-1 and these signaling pathways are consistent with associa-
tions between Cav-1 and the signaling pathways in LPS-induced
chorioamnionitis. This study contains the limitation that a direct
mechanistic linkage between Cav-1 and these signaling pathways
was not shown because of the nature of translational research
Figure 7. Intra-amniotic exposure to LPS induces expression of heme
oxygenase–1 (HO-1). (A) Western blot measurements of HO-1 protein
expression with an anti–HO-1 antibody. The same membrane was an-
alyzed with anti–b-actin antibody. HO-1 and b-actin protein levels were
semiquantified by densitometry. (B) Optical density of HO-1 protein
bands was corrected for b-actin, and results are expressed as ratio (%)
of endotoxin-exposed to control animals. (C–E) Evaluation of HO-1
expression in lung tissue by immunohistochemistry. Sections are rep-
resentative of a control animal (C) and an animal exposed to endo-
toxin-induced chorioamnionitis for 7 days (D), stained for HO-1.
Magnification, 350. (E) Immunohistochemical semiquantification of
HO-1 in lung sections, on a scale from 0–3. Values represent means 6
SE. *P , 0.05, versus control group.
974AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 452011
A next step involves testing therapies that increase the bioavail-
onstrated that the interaction between Cav-1 and the TGF-b
identified as the caveolin scaffolding domain (CSD), which spe-
cifically recognizes and binds to a short amino-acid sequence,
(55). The function of Cav-1 may be mimicked by the delivery of
a penetrating CSD fusion peptide, leading to an inhibition of
TGF-b–induced Smad2/3 activation and collagen expression
(56). This peptide might restore the function of Cav-1 in the set-
ting of lung injury induced by LPS-induced chorioamnionitis.
In conclusion, our study supports a role for Cav-1 in the lung
remodeling induced by antenatal inflammation (Fig. S1 in the
online data supplement). The expression of Cav-1 mRNA and
protein were low in lung tissues after antenatal inflammation.
In contrast, TGF-b1 increased considerably with antenatal in-
flammation–induced lung remodeling (25), providing a basis for
the hypothesis that TGF-b1 may be one of the negative regu-
lators of pulmonary Cav-1 expression (26–28). In addition, the
expression of Cav-1 is associated with the activation of other
signaling pathways and enzymes in the lung. The Stat and
a-SMase/ceramide pathways and the expression of HO-1 may
contribute to inflammation and remodeling in the preterm in-
jured lung. We do not know whether these events are the causes
or effects of the loss of pulmonary Cav-1. Whether Cav-1 exerts
positive or negative effects on airway remodeling also remains
Author Disclosure: None of the authors has a financial relationship with a com-
mercial entity that has an interest in the subject of this manuscript.
Acknowledgments: The authors thank B. Ottensmeier, D. Herbst, and M. Kapp for
excellent technical work.
1. Thomas W, Speer CP. Chorioamnionitis: important risk factor or innocent
bystander for neonatal outcome? Neonatology 2010;99:177–187.
2. Kramer BW, Kallapur S, Newnham J, Jobe AH. Prenatal inflammation
and lung development. Semin Fetal Neonatal Med 2009;14:2–7.
3. Lahra MM, Jeffery HE. A fetal response to chorioamnionitis is associ-
ated with early survival after preterm birth. Am J Obstet Gynecol
4. Yoon BH, Jun JK, Romero R, Park KH, Gomez R, Choi JH, Kim IO.
Amniotic fluid inflammatory cytokines (interleukin-6, interleukin-
1beta, and tumor necrosis factor–alpha), neonatal brain white
matter lesions, and cerebral palsy. Am J Obstet Gynecol 1997;177:
5. Gantert M, Been JV, Gavilanes AW, Garnier Y, Zimmermann LJ,
Kramer BW. Chorioamnionitis: a multiorgan disease of the fetus? J
6. Jobe AJ. The new BPD: an arrest of lung development. Pediatr Res 1999;
7. Razani B, Engelman JA, Wang XB, Schubert W, Zhang XL, Marks CB,
Macaluso F, Russell RG, Li M, Pestell RG, et al. Caveolin-1 null mice
are viable but show evidence of hyperproliferative and vascular ab-
normalities. J Biol Chem 2001;276:38121–38138.
8. Jin Y, Lee SJ, Minshall RD, Choi AM. Caveolin-1: a critical regulator of
lung injury. Am J Physiol Lung Cell Mol Physiol 2011;300:L151–L160.
9. Williams TM, Lisanti MP. The caveolin genes: from cell biology to
medicine. Ann Med 2004;36:584–595.
10. Gosens R, Mutawe M, Martin S, Basu S, Bos ST, Tran T, Halayko AJ.
Caveolae and caveolins in the respiratory system. Curr Mol Med 2008;
11. Razani B, Zhang XL, Bitzer M, von Gersdorff G, Bottinger EP, Lisanti
MP. Caveolin-1 regulates transforming growth factor (TGF)–beta/
SMAD signaling through an interaction with the TGF-beta Type I
receptor. J Biol Chem 2001;276:6727–6738.
12. Santibanez JF, Blanco FJ, Garrido-Martin EM, Sanz-Rodriguez F, del
Pozo MA, Bernabeu C. Caveolin-1 interacts and cooperates with the
transforming growth factor–beta Type I receptor ALK1 in endothelial
caveolae. Cardiovasc Res 2008;77:791–799.
13. Bennett D, Alphey L. PP1 binds Sara and negatively regulates Dpp
signaling in Drosophila melanogaster. Nat Genet 2002;31:419–423.
14. Jasmin JF, Mercier I, Hnasko R, Cheung MW, Tanowitz HB, Dupuis J,
Lisanti MP. Lung remodeling and pulmonary hypertension after
myocardial infarction: pathogenic role of reduced caveolin expression.
Cardiovasc Res 2004;63:747–755.
15. Sehgal PB, Guo GG, Shah M, Kumar V, Patel K. Cytokine signaling:
STATS in plasma membrane rafts. J Biol Chem 2002;277:12067–
16. Veldman RJ, Maestre N, Aduib OM, Medin JA, Salvayre R, Levade T.
A neutral sphingomyelinase resides in sphingolipid-enriched micro-
domains and is inhibited by the caveolin-scaffolding domain: potential
implications in tumour necrosis factor signalling. Biochem J 2001;355:
17. Yang Y, Yin J, Baumgartner W, Samapati R, Solymosi EA, Reppien E,
Kuebler WM, Uhlig S. Platelet-activating factor reduces endothelial
nitric oxide production: role of acid sphingomyelinase. Eur Respir J
18. Gao H, Ward PA. STAT3 and suppressor of cytokine signaling 3: po-
tential targets in lung inflammatory responses. Expert Opin Ther
19. Severgnini M, Takahashi S, Rozo LM, Homer RJ, Kuhn C, Jhung JW,
Perides G, Steer M, Hassoun PM, Fanburg BL, et al. Activation of the
STAT pathway in acute lung injury. Am J Physiol Lung Cell Mol
20. Severgnini M, Takahashi S, Tu P, Perides G, Homer RJ, Jhung JW,
Bhavsar D, Cochran BH, Simon AR. Inhibition of the Src and Jak
kinases protects against lipopolysaccharide-induced acute lung injury.
Am J Respir Crit Care Med 2005;171:858–867.
21. Uhlig S, Gulbins E. Sphingolipids in the lungs. Am J Respir Crit Care
22. von Bismarck P, Wistadt CF, Klemm K, Winoto-Morbach S, Uhlig U,
Schutze S, Adam D, Lachmann B, Uhlig S, Krause MF. Improved
pulmonary function by acid sphingomyelinase inhibition in a new-
born piglet lavage model. Am J Respir Crit Care Med 2008;177:
23. Jin Y, Kim HP, Chi M, Ifedigbo E, Ryter SW, Choi AM. Deletion of
caveolin-1 protects against oxidative lung injury via up-regulation of
heme oxygenase–1. Am J Respir Cell Mol Biol 2008;39:171–179.
24. Fredenburgh LE, Perrella MA, Mitsialis SA. The role of heme oxy-
genase–1 in pulmonary disease. Am J Respir Cell Mol Biol 2007;36:
25. Kunzmann S, Speer CP, Jobe AH, Kramer BW. Antenatal inflammation
induced TGF-beta1 but suppressed CTGF in preterm lungs. Am J
Physiol Lung Cell Mol Physiol 2007;292:L223–L231.
26. Ding H, Zhou FQ, Cai HR, Zhou YH, Meng FQ. Expression of
caveolin-1 and extracellular matrix induced by transforming growth
factor beta1 in human fetal lung fibroblasts [in Chinese]. Zhonghua
Jie He He Hu Xi Za Zhi 2010;33:280–283.
27. Igarashi J, Shoji K, Hashimoto T, Moriue T, Yoneda K, Takamura T,
Yamashita T, Kubota Y, Kosaka H. Transforming growth factor–
beta1 downregulates caveolin-1 expression and enhances sphingosine
1-phosphate signaling in cultured vascular endothelial cells. Am J
Physiol Cell Physiol 2009;297:C1263–C1274.
28. Wang XM, Zhang Y, Kim HP, Zhou Z, Feghali-Bostwick CA, Liu F,
Ifedigbo E, Xu X, Oury TD, Kaminski N, et al. Caveolin-1: a critical
regulator of lung fibrosis in idiopathic pulmonary fibrosis. J Exp Med
29. Kramer BW, Moss TJ, Willet KE, Newnham JP, Sly PD, Kallapur SG,
Ikegami M, Jobe AH. Dose and time response after intraamniotic
endotoxin in preterm lambs. Am J Respir Crit Care Med 2001;164:
30. Kramer BW, Kallapur SG, Moss TJ, Nitsos I, Newnham JP, Jobe AH.
Intra-amniotic LPS modulation of TLR signaling in lung and blood
monocytes of fetal sheep. Innate Immun 2009;15:101–107.
Kunzmann, Collins, Yang, et al.: Regulation of Cav-1 by Inflammation in Preterm Fetal Lungs975
31. Kramer BW, Kramer S, Ikegami M, Jobe AH. Injury, inflammation, and Download full-text
remodeling in fetal sheep lung after intra-amniotic endotoxin. Am J
Physiol Lung Cell Mol Physiol 2002;283:L452–L459.
32. Wiegmann K, Schutze S, Machleidt T, Witte D, Kronke M. Functional
dichotomy of neutral and acidic sphingomyelinases in tumor necrosis
factor signaling. Cell 1994;78:1005–1015.
33. Jensen JM, Schutze S, Forl M, Kronke M, Proksch E. Roles for tumor
necrosis factor receptor p55 and sphingomyelinase in repairing the
cutaneous permeability barrier. J Clin Invest 1999;104:1761–1770.
34. Benjamin JT, Carver BJ, Plosa EJ, Yamamoto Y, Miller JD, Liu JH, van
der Meer R, Blackwell TS, Prince LS. NF-kappaB activation limits
airway branching through inhibition of Sp1-mediated fibroblast
growth factor–10 expression. J Immunol 2010;185:4896–4903.
35. Benjamin JT, Gaston DC, Halloran BA, Schnapp LM, Zent R, Prince
LS. The role of integrin alpha8beta1 in fetal lung morphogenesis and
injury. Dev Biol 2009;335:407–417.
36. Kunzmann S, Glogger K, Been JV, Kallapur SG, Nitsos I, Moss TJ,
Speer CP, Newnham JP, Jobe AH, Kramer BW. Thymic changes after
chorioamnionitis induced by intraamniotic lipopolysaccharide in fetal
sheep. Am J Obstet Gynecol 2010;202:e471–e479.
37. Moss TJ, Newnham JP, Willett KE, Kramer BW, Jobe AH, Ikegami M.
Early gestational intra-amniotic endotoxin: lung function, surfactant,
and morphometry. Am J Respir Crit Care Med 2002;165:805–811.
38. Wolfs TG, Buurman WA, Zoer B, Moonen RM, Derikx JP, Thuijls G,
Villamor E, Gantert M, Garnier Y, Zimmermann LJ, et al. Endotoxin
induced chorioamnionitis prevents intestinal development during
gestation in fetal sheep. PLoS ONE 2009;4:e5837.
39. Jobe AH, Newnham JP, Willet KE, Moss TJ, Gore Ervin M, Padbury JF,
Sly P, Ikegami M. Endotoxin-induced lung maturation in preterm
lambs is not mediated by cortisol. Am J Respir Crit Care Med 2000;
40. Kallapur SG, Moss TJ, Auten RL Jr, Nitsos I, Pillow JJ, Kramer BW,
Maeda DY, Newnham JP, Ikegami M, Jobe AH. IL-8 signaling does
not mediate intra-amniotic LPS-induced inflammation and matura-
tion in preterm fetal lamb lung. Am J Physiol Lung Cell Mol Physiol
41. Kallapur SG, Nitsos I, Moss TJ, Polglase GR, Pillow JJ, Cheah FC, Kramer
BW, Newnham JP, Ikegami M, Jobe AH. IL-1 mediates pulmonary and
systemic inflammatory responses to chorioamnionitis induced by lipo-
polysaccharide. Am J Respir Crit Care Med 2009;179:955–961.
42. DiGiulio DB, Romero R, Amogan HP, Kusanovic JP, Bik EM, Gotsch F,
Kim CJ, Erez O, Edwin S, Relman DA. Microbial prevalence, diversity
and abundance in amniotic fluid during preterm labor: a molecular and
culture-based investigation. PLoS ONE 2008;3:e3056.
43. Kotecha S, Wangoo A, Silverman M, Shaw RJ. Increase in the con-
centration of transforming growth factor beta–1 in bronchoalveolar
lavage fluid before development of chronic lung disease of pre-
maturity. J Pediatr 1996;128:464–469.
44. Lecart C, Cayabyab R, Buckley S, Morrison J, Kwong KY, Warburton
D, Ramanathan R, Jones CA, Minoo P. Bioactive transforming
growth factor–beta in the lungs of extremely low birthweight neonates
predicts the need for home oxygen supplementation. Biol Neonate
45. Del Galdo F, Lisanti MP, Jimenez SA. Caveolin-1, transforming growth
factor–beta receptor internalization, and the pathogenesis of systemic
sclerosis. Curr Opin Rheumatol 2008;20:713–719.
46. Di Guglielmo GM, Le Roy C, Goodfellow AF, Wrana JL. Distinct
endocytic pathways regulate TGF-beta receptor signalling and turn-
over. Nat Cell Biol 2003;5:410–421.
47. Le Saux O, Teeters K, Miyasato S, Choi J, Nakamatsu G, Richardson
JA, Starcher B, Davis EC, Tam EK, Jourdan-Le Saux C. The role of
caveolin-1 in pulmonary matrix remodeling and mechanical proper-
ties. Am J Physiol Lung Cell Mol Physiol 2008;295:L1007–L1017.
48. Le Saux CJ, Teeters K, Miyasato SK, Hoffmann PR, Bollt O, Douet V,
Shohet RV, Broide DH, Tam EK. Down-regulation of caveolin-1, an
inhibitor of transforming growth factor–beta signaling, in acute
allergen-induced airway remodeling. J Biol Chem 2008;283:5760–
49. Darnell JE Jr. STATs and gene regulation. Science 1997;277:1630–1635.
50. Park DS, Lee H, Frank PG, Razani B, Nguyen AV, Parlow AF, Russell
RG, Hulit J, Pestell RG, Lisanti MP. Caveolin-1–deficient mice show
accelerated mammary gland development during pregnancy, pre-
mature lactation, and hyperactivation of the Jak-2/STAT5a signaling
cascade. Mol Biol Cell 2002;13:3416–3430.
51. Marchesini N, Hannun YA. Acid and neutral sphingomyelinases: roles
and mechanisms of regulation. Biochem Cell Biol 2004;82:27–44.
52. Kim HP, Wang X, Galbiati F, Ryter SW, Choi AM. Caveolae com-
partmentalization of heme oxygenase–1 in endothelial cells. FASEB J
53. Farkas I, Maroti Z, Katona M, Endreffy E, Monostori P, Mader K, Turi
S. Increased heme oxygenase–1 expression in premature infants with
respiratory distress syndrome. Eur J Pediatr 2008;167:1379–1383.
54. Maroti Z, Katona M, Orvos H, Nemeth I, Farkas I, Turi S. Heme oxy-
genase–1 expression in premature and mature neonates during the
first week of life. Eur J Pediatr 2007;166:1033–1038.
55. Razani B, Lisanti MP. Caveolin-deficient mice: insights into caveolar
function human disease. J Clin Invest 2001;108:1553–1561.
56. Tourkina E, Richard M, Gooz P, Bonner M, Pannu J, Harley R,
Bernatchez PN, Sessa WC, Silver RM, Hoffman S. Antifibrotic
properties of caveolin-1 scaffolding domain in vitro and in vivo. Am J
Physiol Lung Cell Mol Physiol 2008;294:L843–L861.
976AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 452011