Wild-type levels of the mouse Forkhead Box f1 gene
are essential for lung repair
Vladimir V.
Kalinichenko1,
Yan
Zhou1,
Brian
Shin1,
Donna Beer
Stolz2,
Simon C.
Watkins2,
Jeffrey A.
Whitsett3, and
Robert H.
Costa1
1 Department of Molecular Genetics, College of
Medicine, University of Illinois at Chicago, Chicago, Illinois
60607-7170; 2 Center for Biologic Imaging, University of
Pittsburgh, Pittsburgh, Pennsylvania 15261; and
3 Division of Pulmonary Biology, Children's
Hospital Medical Center, Cincinnati, Ohio 45229-3039
 |
ABSTRACT |
The Forkhead
Box (Fox) family of transcription factors plays important roles in
regulating expression of genes involved in cellular proliferation and
differentiation. In a previous study, we showed that newborn
foxf1(+/
) mice with diminished Foxf1 levels exhibited
abnormal formation of pulmonary alveoli and capillaries and died
postnatally. Interestingly, surviving newborn foxf1(+/
) mice exhibited increased pulmonary Foxf1 levels and normal adult lung
morphology, suggesting that wild-type Foxf1 levels are required for
lung development and function. The present study was conducted to
determine whether adult foxf1(+/
) mice were able to
undergo lung repair similar to that observed in wild-type mice. We
demonstrated that adult foxf1(+/
) mice died from severe
lung hemorrhage after butylated hydroxytoluene (BHT) lung injury and
that this phenotype was associated with a 10-fold decrease in pulmonary
Foxf1 expression and increased alveolar endothelial cell
apoptosis that disrupted capillary integrity. Furthermore,
BHT-induced lung hemorrhage of adult foxf1(+/
) mice was
associated with a drastic reduction in expression of the Flk-1, bone
morphogenetic protein-4, surfactant protein B, platelet endothelial
cell adhesion molecule, and vascular endothelial cadherin genes,
whereas the expression of these genes was either transiently diminished
or increased in wild-type lungs after BHT injury. Because these
proteins are critical for lung morphogenesis and endothelial
homeostasis, their decreased mRNA levels are likely contributing to
BHT-induced lung hemorrhage in foxf1(+/
) mice.
Collectively, our data suggest that sustained expression of Foxf1 is
essential for normal lung repair and endothelial cell survival in
response to pulmonary cell injury.
winged helix deoxyribonucleic acid-binding domain; Flk-1; bone morphogenetic protein-4; surfactant protein B; platelet
endothelial cell adhesion molecule-1; vascular endothelial cadherin; butylated hydroxytoluene lung injury; endothelial cells
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INTRODUCTION |
THE FORKHEAD
BOX (Fox) family of transcription factors (20)
shares homology in the winged helix DNA-binding domain
(6), and its members play important roles in regulating
transcription of genes involved in cellular proliferation,
differentiation, and metabolic homeostasis (9, 10, 19, 24, 25,
37, 42, 48). One of these family members, Foxf1 (also known as HFH-8 or Freac-1), initiates expression during gastrulation in a subset
of mesodermal cells arising from the primitive streak region that
contributes to the extraembryonic mesoderm and lateral mesoderm
(38). Consistent with this early expression pattern, foxf1(
/
) embryos die in utero from defects in
extraembryonic and lateral mesoderm differentiation (29).
During organogenesis, high levels of Foxf1 expression persist in the
mesenchyme of the respiratory and gastrointestinal tracts. Foxf1 RNA is
expressed at mesenchymal-epithelial interfaces involved in lung and gut morphogenesis (30, 38). In the adult mouse, Foxf1 RNA is
detected in smooth muscle layers of pulmonary bronchioles, lamina
propria of the stomach and the intestine, and in alveolar endothelial cells (22, 38).
A study conducted in our laboratory demonstrated that ~55% of
newborn foxf1(+/
) mice died of severe lung
hemorrhage (21), and a study conducted in another
laboratory confirmed perinatal lethality of
foxf1(+/
) mice using a different genetic background (28). The lethal phenotype was associated with
foxf1(+/
) lungs displaying an 80% reduction in Foxf1 mRNA
levels compared with wild-type lungs [designated as low
foxf1(+/
) mice; see Ref. 21]. Low
foxf1(+/
) newborn mice exhibited defects in formation of pulmonary capillaries and alveoli with disruption of the
mesenchymal-epithelial cell interfaces and increased apoptosis
in the alveolar and bronchiolar regions of the lung parenchyma
(21). Furthermore, lung hemorrhage was associated
with reduced expression of the vascular endothelial growth factor
(VEGF) receptor 2 (Flk-1), platelet endothelial cell adhesion
molecule-1 (PECAM-1), surfactant protein B (SP-B), and bone
morphogenetic protein-4 (BMP-4) as well as the lung Kruppel-like factor
and T-box (Tbx2-Tbx5) transcription factors.
Interestingly, expression of these genes was unchanged in 40% of the
newborn foxf1(+/
) mice that possessed wild-type pulmonary
levels of Foxf1 mRNA [high foxf1(+/
) mice] but exhibited
diminished alveolar septation without pulmonary hemorrhage
(21). Moreover, the high foxf1(+/
) mice had
normal life spans and adult lung morphology, suggesting that
compensation for developmental defects in septation had occurred.
Butylated hydroxytoluene (BHT)-mediated lung injury is characterized
initially by extensive damage to the distal lung epithelial and
endothelial cells and subsequently by cellular proliferation between 3 and 7 days after BHT administration (1, 31). The cellular
repair process after BHT lung injury is associated with a transient
65% reduction in pulmonary Foxf1 mRNA levels between 4 and 6 days
after BHT injury and a return to normal levels by day 8 (22). In this study, we used BHT lung injury to examine whether adult foxf1(+/
) mice are capable of normal lung
repair in response to cellular damage. Within 7 days after BHT lung
injury, all foxf1(+/
) mice died from severe lung
hemorrhage. BHT-mediated induction of foxf1(+/
) pulmonary
hemorrhage was associated with a 10-fold decrease in pulmonary Foxf1
expression and increased apoptosis of alveolar endothelial
cells that were positive for Foxf1, PECAM-1, and CD34 expression.
Furthermore, the severe foxf1(+/
) pulmonary phenotype
after BHT injury was associated with reduced expression of Flk-1,
vascular endothelial (VE) cadherin, BMP-4, SP-B, and PECAM-1, all
of which are required for lung morphogenesis and endothelial cell homeostasis.
 |
MATERIALS AND METHODS |
foxf1(+/
) mice and BHT
treatment.
The generation of mice heterozygous for a targeted deletion of the
Foxf1 locus has been described previously, and foxf1(+/
) mice were maintained in the 129/black Swiss mouse background
(21). The winged helix DNA-binding domain of Foxf1 was
replaced by an in-frame insertion of a nuclear localizing
-galactosidase (
-Gal) gene, disrupting function of the mouse gene
in vivo (21). Expression of the
-Gal gene was therefore
under the control of Foxf1 regulatory sequences, thus allowing the use
of
-Gal enzyme staining for visualizing the expression pattern of
Foxf1. Tail tissue samples were used to prepare genomic DNA for
genotyping of the newborn mice by PCR analysis, as described previously
(21).
BHT (3,5-di-t-butyl-4-hydroxytoluene; Sigma, St. Louis, MO)
was dissolved in corn oil (Mazola) at a concentration 30 mg/ml, and a
single intraperitoneal injection of BHT (300 mg/kg body wt) was given
to foxf1(+/
) mice or their wild-type littermates (22). To determine statistical significance of any
observed differences, we used three mice per time point after BHT
administration, which included 2, 3, 4, 6, and 8 days. The mice were
killed by CO2 asphyxiation, and lung tissue was weighed and
used to prepare total RNA, or lungs were inflated with 4%
paraformaldehyde (PFA), fixed overnight in 4% PFA at 4°C, and then
paraffin embedded as described previously (21, 22).
Immunohistochemical staining, TdT-UTP nick end-labeling
apoptosis assay and transmission electron microscopy.
Sections of paraffin-embedded lung tissue were stained with hematoxylin
and eosin or used for immunohistochemical staining with the following
rat monoclonal antibodies: PECAM-1 (clone MEC 13.3) and CD34
(clone RAM34) both from Pharmingen (San Diego, CA). Antibody-antigen
complexes were detected using biotinylated horse anti-rat antibody and
avidin-alkaline phosphatase complex with 5-bromo-4-chloro-3-indolyl
phosphate (BCIP)/nitro blue tetrazolium (NBT) substrate and then were
counterstained with nuclear fast red (Vector Laboratories, Burlingame,
CA) as described previously (21). We also used mouse
monoclonal antibodies against
-smooth muscle actin (clone 1A4;
Sigma) and proliferation cell nuclear antigen (PCNA, clone PC10; Roche
Molecular Biochemicals, Indianapolis, IN) and detected the
antibody-antigen complex using horse anti-mouse antibody conjugated
with alkaline phosphatase (Vector Laboratories), as described
previously (21). To measure apoptosis in wild-type and foxf1(+/
) mice at various intervals after BHT lung
injury (0, 4, and 6 days), TdT-UTP nick end-labeling (TUNEL) assay was performed using an in situ cell death detection kit from Roche Diagnostic according to the manufacturer's recommendations.
Apoptotic cells were visualized using BCIP/NBT substrate for
alkaline phosphatase and then were counterstained with nuclear fast red
(21). We counted apoptotic or PCNA-positive cells from
five ×200 lung viewing fields for each of three mice to determine the
mean and used this to calculate the mean number of apoptotic cells
(±SD) for three mice.
Adult lung tissue was isolated from foxf1(+/
) or wild-type
mice at 4 days after BHT lung injury, PFA fixed, and prepared for
transmission electron microscopy (TEM), as described previously (39). Lung tissue sections were photographed using a Jeol
(Peabody, MA) JEM 1210 transmission electron microscope at 80 or 60 kV
on electron microscope film (ESTAR thick base; Kodak, Rochester, NY)
and printed on photographic paper.
-Gal enzyme staining.
Staining for expression of
-Gal was performed according to
Clevidence et al. (7), with a few modifications. Briefly,
dissected lungs were fixed for 1 h in PBS (pH 7.8) containing 2%
formaldehyde-0.2% glutaraldehyde, 0.02% Nonidet P-40 (NP-40), and
0.01% sodium deoxycholate. Lung tissue was then stained for
-Gal
enzyme activity in a PBS solution containing 5 mM potassium
ferrocyanide, 5 mM ferricyanide, 2 mM magnesium chloride, 0.02% NP-40,
0.01% sodium deoxycholate, and 1 mg/ml
5-bromo-4-chloro-3-indolyl-
-D-galactoside for 4-6 h
at 37°C. After
-Gal staining, samples were rinsed three times in
PBS, postfixed overnight in 4% PFA, and then paraffin embedded (21). For colocalization studies,
-Gal-stained sections
were treated with xylene to remove paraffin wax, rehydrated, and used for TUNEL assay to measure apoptosis.
RNA extraction and RNase protection assay.
Total mouse lung RNA was prepared by an acid
guanidium-thiocyanate-phenol-chloroform extraction method using
RNA-STAT-60 (Tel-Test "B," Friendswood, TX). RNase protection assay
was performed with [32P]UTP-labeled antisense RNA
synthesized from plasmid templates with the appropriate RNA polymerase,
as described previously (8). RNA probe hybridization,
RNase One (Promega, Madison, WI) digestion, electrophoresis of
RNA-protected fragments, and autoradiography were performed as
described previously (22, 39, 41, 49). Quantitation of
expression levels was determined from scanned X-ray films by using the
BioMax 1D program (Kodak). The cyclophilin hybridization signal was
used for normalization control between different lung RNA samples.
Synthesis of antisense mouse Foxf1, Flk-1, PECAM-1, VEGF, BMP-4,
FoxM1B, cyclophilin, and rat SP-B RNA probes was described previously
(21, 22, 41). Antisense RNA probes for VE cadherin, heme
oxygenase 1, and vimentin were generated from Atlas cDNA plasmids
purchased from Clontech (Palo Alto, CA). The expression of bcl-w and
interleukin (IL)-6 genes was determined using mouse Apo-2 and mCK-2
templates from Pharmingen. The Foxf2 RNase protection probe was
generated by PCR amplification of mouse genomic DNA using the following
primers: 5'-gcggaattccctgacctcaagcagccg and
5'-gcgggattccagccttggcgctcttta. The resulting Foxf2 genomic PCR
fragment was cloned into BlueScript plasmid (Stratagene) as an
EcoRI and BamHI fragment, and T3 RNA polymerase
was used to synthesize Foxf2 antisense RNA probe from a
ClaI-digested template.
 |
RESULTS |
BHT lung injury induces pulmonary hemorrhage in
foxf1(+/
) mice.
Hematoxylin and eosin staining of adult lung sections showed that the
foxf1(+/
) pulmonary morphology was indistinguishable from
the wild type (Fig. 1, A and
B). We therefore used BHT lung injury to examine whether
adult foxf1(+/
) mice displayed a normal lung repair
process. Adult foxf1(+/
) mice and their wild-type littermates were injected with BHT, and their lungs were harvested at
intervals after BHT injury. Although wild-type mice exhibited only a
single mortality after BHT lung injury, none of the 16 foxf1(+/
) mice survived >7 days after BHT administration
(Fig. 2A). By 3 days after BHT
injury, the lung weight-to-body weight ratio was significantly
increased in foxf1(+/
) mice compared with wild-type
controls (Fig. 2B), suggesting an accumulation of
extracellular fluid in foxf1(+/
) lung tissue. At 4 days
post-BHT lung injury, both wild-type and foxf1(+/
) lungs
displayed visible lung damage with alveolar infiltration of
inflammatory cells, but hemorrhage was seen only in lungs from
foxf1(+/
) mice (Fig. 1, C-E). By day
6 after BHT injury, histological improvement was noted in the
lungs of wild-type mice (Fig. 1F), whereas
foxf1(+/
) lungs exhibited severe pulmonary hemorrhage
(Fig. 1, G and H). These results suggest that the
foxf1(+/
) mice died from severe pulmonary hemorrhage
induced by BHT injury.

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Fig. 1.
Butylated hydroxytoluene (BHT) injury of Forkhead Box
(Fox) f1(+/ ) mice causes pulmonary hemorrhage.
A-H: BHT lung injury induces pulmonary hemorrhage in
foxf1(+/ ) mice. Paraffin sections from adult wild-type
(A, C, and F) and
foxf1(+/ ) lungs (B, D, E,
G, and H) were stained with hematoxylin and
eosin. In untreated mice, wild-type (A) and
foxf1(+/ ) (B) lungs show similar morphology.
Pulmonary hemorrhage (alveolar red blood cells) was noted in
foxf1(+/ ) mice by day 4 after BHT lung injury
(D and E), and severe pulmonary hemorrhage was
noted at day 6 (G and H). No pulmonary
hemorrhage was detected in wild-type (wt) mice after BHT lung injury
(C and F). Magnification: A-D,
F, and G are at ×200; E and
H are at ×600.
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Fig. 2.
BHT treatment of foxf1(+/ ) mice causes
mortality and increase in lung weight. A: graphic
representation of percentage of surviving wild-type and
foxf1(+/ ) mice after BHT lung injury. Wt and
foxf1(+/ ) mice were injected with BHT, and percentage of
survival at different intervals after BHT lung injury was calculated.
B: foxf1(+/ ) mice exhibit increase in ratio of
lung weight to body weight in response to BHT lung injury. Mice were
injected with BHT, lungs were harvested at different time points after
BHT administration, and freshly dissected lung tissue was weighed. The
ratio of lung weight (mg) to body weight (g) was calculated from 3 different mice and presented as the mean ± SD.
* P < 0.05, statistically significant differences
between foxf1(+/ ) and wt lungs.
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foxf1(+/
) pulmonary
hemorrhage is associated with significant decreases in pulmonary
expression of Foxf1 mRNA.
We recently reported that a subset of newborn foxf1(+/
)
mice exhibited lethal pulmonary hemorrhage, which correlated with diminished pulmonary expression of Foxf1 mRNA (21). The
surviving foxf1(+/
) mice displayed wild-type pulmonary
levels of Foxf1 expression (Fig. 3,
A-C), suggesting that normal Foxf1 levels are necessary
for appropriate lung morphogenesis and function (21). To
determine whether BHT-induced lung hemorrhage was associated with
decreased Foxf1 expression, RNase protection assays were performed to
measure pulmonary Foxf1 mRNA levels. At 2 days after BHT lung injury,
foxf1(+/
) mice displayed a more precipitous 70% decline
in pulmonary Foxf1 mRNA than the 20% reduction noted in wild-type
lungs (Fig. 3, A-C). Both foxf1(+/
) and
wild-type mice displayed a similar reduction in pulmonary Foxf1 mRNA
between days 3 and 4 post-BHT lung injury (Fig.
3, A-C). Although wild-type mice exhibited increased
pulmonary expression of Foxf1 by day 6 after BHT lung
injury, foxf1(+/
) lungs exhibited further decreases in
Foxf1 mRNA (Fig. 3, B and C). This 90% reduction
in pulmonary Foxf1 mRNA was associated with severe lung hemorrhage and
mortality in foxf1(+/
) mice at 6 days after BHT lung
injury.

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Fig. 3.
BHT lung injury significantly reduces Foxf1 and Foxf2 mRNA
expression in foxf1(+/ ) lungs. Total RNA was prepared from
wt (A) and foxf1(+/ ) (B) mice at
various intervals after BHT lung injury and analyzed for Foxf1, Foxf2,
and cyclophilin (Cycl) mRNA by RNase protection assay. Numbers at
bottom in A and B represent averages
of pulmonary mRNA levels in mice treated with BHT compared with
untreated controls. Graphic representation of Foxf1 (C) and
Foxf2 (D) mRNA levels after BHT lung injury. Each individual
sample was normalized to its corresponding cyclophilin levels and
expressed as the degree of induction compared with wt lungs (untreated
set at 1.0). Mean degree of induction ± SD was calculated from 3 different mice. E: relative number of -galactosidase
( -Gal)-positive nuclei was calculated using three different
foxf1(+/ ) mice and was plotted as a mean of the percentage
of -Gal-positive cells ± SD.
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Because of the similarities between Foxf1 and Foxf2 (Freac-2, Lun)
proteins in expression pattern and the winged helix DNA-binding sequence (2, 16, 17, 30, 33, 38), we next examined Foxf2
mRNA levels in BHT-injured lungs (Fig. 3D). Wild-type lungs exhibited a transient 60% reduction in Foxf2 mRNA between 2 and 4 days
after BHT injury, and its expression recovered by day 6 (Fig. 3, A and D). As was the case with Foxf1
expression, BHT-injured foxf1(+/
) lungs exhibited a
further decrease in Foxf2 mRNA at day 6 (Fig. 3,
A-D). This result suggests that, in
foxf1(+/
) lungs, BHT did not induce Foxf2 mRNA to
compensate for the precipitous decline in Foxf1 expression.
Because the targeted Foxf1 allele possesses an in-frame insertion of
the nuclear localizing
-Gal gene, staining for
-Gal enzyme
activity allows the identification of Foxf1-expressing cells (21,
22). We next stained foxf1(+/
) lungs for
-Gal enzyme activity at different intervals after BHT injury to determine whether the lung damage caused a reduction in the number of
Foxf1-expressing cells (Fig. 4,
A-D). In untreated foxf1(+/
) lung tissue,
-Gal-positive cells represent ~52% of the total cells in the lung
parenchyma (Fig. 3E). However, the foxf1(+/
)
alveolar region exhibited a 50% decrease in
-Gal-positive cells by
6 days after BHT treatment (Figs. 3E and 4,
A-D). In contrast, no decrease in
-Gal-positive peribronchiolar smooth muscle cells was noted after BHT lung injury (Fig. 4, E-F). Consistent with this result,
BHT-injured foxf1(+/
) lungs display unchanged
expression of
-smooth muscle actin in the peribronchiolar smooth
muscle cells (Fig. 4, I-J), suggesting that these
Foxf1-expressing cells were not severely affected by BHT lung injury.

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Fig. 4.
BHT lung injury diminishes the number of alveolar Foxf1-expressing
cells in foxf1(+/ ) lungs but does not alter proliferation.
Because the targeted Foxf1 allele possess an in-frame insertion of
nuclear-localizing -Gal gene, staining for -Gal enzyme activity
allows the identification of Foxf1-expressing cells (21,
22). To visualize Foxf1-expressing cells, foxf1(+/ )
lung tissue was isolated from mice at various intervals after BHT lung
injury and stained for -Gal enzyme activity with
5-bromo-4-chloro-3-indolyl- -D-galactoside substrate
(blue stains), paraffin embedded, sectioned, and couterstained with
nuclear fast red (red stains) as described in MATERIALS AND
METHODS. A-D: BHT injury of
foxf1(+/ ) lungs caused diminished number of alveolar
Foxf1-expressing endothelial cells, as assessed by staining for -Gal
enzyme activity (blue stains, some of which are indicated by arrows).
BHT injury of foxf1(+/ ) lungs did not change the number of
peribronchiolar smooth muscle cells, as assessed by staining for
-Gal enzyme activity (E and F) and
immunohistochemical staining for -smooth muscle actin ( -SMA)
protein (I and J). K and L:
TdT-UTP nick end-labeling (TUNEL) staining for apoptotic (Apop)
cells (brown stains) colocalize with -Gal-positive cells (blue
stains, Foxf1-expressing cells) in alveolar region and are
indicated by arrows. Magnification:
A-D are at ×400; E-J
are at ×200; K and L are at ×600. Br,
bronchiolar; Ar, artery.
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BHT-injured foxf1(+/
) lungs
exhibit an increase in apoptosis and persistent proliferation.
Because BHT injury of foxf1(+/
) lungs caused a decline in
alveolar Foxf1-expressing endothelial cells, we next used TUNEL assay
to examine whether the foxf1(+/
) lungs displayed an
increase in apoptosis. Although BHT injury induced visible
apoptosis in wild-type lungs (Figs.
5A and
6, A, C, and
E), a fivefold increase in the number of apoptotic cells
was observed throughout the alveolar region of foxf1(+/
)
lungs (Figs. 5A and 6, B, D, and
F). Colocalization studies showed that the majority of
BHT-induced apoptotic cells were the
-Gal-positive
(Foxf1-expressing) endothelial cells of the pulmonary alveolar region
(Fig. 4, K-L). These results suggest that increased
alveolar endothelial cell apoptosis may contribute to the
observed lung hemorrhage in BHT-treated foxf1(+/
) mice. However, the TUNEL assay cannot rule out the possibility that necrosis
also occurred in BHT-injured foxf1(+/
) lungs and
contributed to the foxf1(+/
) phenotype.
Immunohistochemical staining with PCNA antibody demonstrated that
wild-type and foxf1(+/
) lungs exhibited equivalent PCNA
staining at 4 days post-BHT treatment (Fig. 5B). Although
PCNA staining had subsided in wild-type lungs by day 6 after
BHT injury, proliferation remained elevated in foxf1(+/
)
lungs (Fig. 5B), perhaps in response to increased alveolar apoptosis (Fig. 5A). In support of this concept,
BHT-injured foxf1(+/
) lungs also displayed a 60-80%
reduction in the expression of the bcl-2 cell survival family member
bcl-w (Fig. 5C). These results correspond with recently
reported data describing a correlation between apoptosis and
differential regulation of bcl-2 family genes after hyperoxia-induced
lung injury (13, 35). Normal induction of the
proliferation-specific FoxM1B transcription factor was found in the
foxf1(+/
) lungs after BHT injury (22, 41, 47), supporting the notion that the global proliferative
response was not impaired in foxf1(+/
) lungs (Fig.
5C). These results suggest that the decline in alveolar
Foxf1-expressing cells in BHT-injured foxf1(+/
) lungs was
the result of increased apoptosis and not diminished cellular
proliferation.

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Fig. 5.
BHT injury of foxf1(+/ ) lungs causes an
increase in apoptosis and proliferation. At various intervals
after BHT lung injury, lung sections were prepared from wt or
foxf1(+/ ) mice and were used for either TUNEL assay
to detect apoptotic cells (Fig. 6) or immunohistochemical
staining with proliferation cell nuclear antigen (PCNA) antibody to
detect DNA replication (data not shown). A: increased
apoptosis in foxf1(+/ ) lungs after BHT lung
injury. Graphic representation of the mean number of apoptotic
cells per ×200 field (±SD for 3 mice) in wt and
foxf1(+/ ) lungs at 0, 4, and 6 days after BHT lung injury,
as described in MATERIALS AND METHODS. B:
foxf1(+/ ) lungs display aberrant PCNA staining at
day 6 after BHT lung injury. Sustained PCNA staining (mean
PCNA-positive cells ± SD) was observed in foxf1(+/ )
lungs compared with wt lungs at 6 days after BHT injury, whereas an
equivalent increase in PCNA staining was observed at 4 days post-BHT
treatment. C: altered expression of apoptotic and
proliferation-promoting genes. Total RNA was prepared from mouse lungs
of wt and foxf1(+/ ) mice at various intervals after BHT
treatment and were analyzed for FoxM1B, bcl-w, and cyclophilin (Cyclo)
mRNA levels by RNase protection assays. FoxM1B expression is induced
during cellular proliferation (22, 41, 42, 47). Each
individual sample was normalized to its corresponding cyclophilin
levels as described in MATERIALS AND METHODS.
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Fig. 6.
BHT injury of foxf1(+/ ) lungs causes an increase in
apoptosis in alveolar region. At various intervals after BHT
lung injury, lung sections were prepared from wt (A,
C, and E) or foxf1(+/ ) mice
(B, D, and F) and were used for TUNEL
assay to detect apoptotic cells. Only sporadic apoptotic cells
(darkly stained, indicated by arrows) were detected in untreated wt
(A) and Foxf1 (B) lungs. Although wt lungs
exhibited a few apoptotic cells after BHT treatment (C
and E), numerous apoptotic cells were detected
throughout the lung parenchyma of BHT-treated foxf1(+/ )
mice (D and F). Magnification: ×200 for
A-F.
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We next used TEM to examine the interface between the alveolar
endothelial cells and type I epithelial cells in wild-type and
foxf1(+/
) lungs at 4 days after BHT lung injury (Fig.
7). Although BHT injury of both wild-type
and foxf1(+/
) lungs caused edema between the two cell
types (Fig. 7, A and B), only
foxf1(+/
) lungs displayed extensive apoptosis of
alveolar endothelial cells and disruption of the capillary wall (Fig.
7, C and D). These TEM studies confirm that BHT
injury of foxf1(+/
) lungs leads to increased
apoptosis of alveolar endothelial cells, causing capillary
damage. These results suggest that Foxf1 plays an important role in
mediating lung morphogenesis and alveolar endothelial cell survival
during cellular repair in response to pulmonary injury.

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Fig. 7.
Transmission electron micrographs of alveolar capillaries
from wt or foxf1(+/ ) lungs after BHT injury.
Ultrastructural analysis of adult and foxf1(+/ )
lungs at 4 days after BHT injury. A and B: BHT
injury of both wt and Foxf1 heterozygous(+/ ) lungs caused edema (*)
around alveolar capillary endothelial cells (EC) containing red blood
cells (RBC). Only foxf1(+/ ) lungs displayed loss of
alveolar endothelium within the capillary wall (arrowheads in
B) and apoptosis of alveolar endothelial cells
(C). Disruption of the capillary wall in
foxf1(+/ ) lung (arrowhead) and neutrophil (N) is breaking
through the alveolar wall in this weakened capillary (D).
T1C, type 1 cell.
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Decreased alveolar PECAM-1 and CD34 staining of endothelial
cells in BHT-injured foxf1(+/
)
lungs.
Previous studies demonstrated that Foxf1 expression colocalizes with
PECAM-1-positive alveolar endothelial cells (22). We therefore used immunohistochemical staining to assess the
pulmonary expression of PECAM-1 in wild-type and
foxf1(+/
) lungs after BHT injury. Before BHT injury,
equivalent alveolar PECAM-1 staining was found in
foxf1(+/
) and wild-type mice (Fig.
8, A and B). Although PECAM-1 staining did not change in BHT-injured wild-type lung
(Fig. 8, C, E, and G), drastic
reductions in alveolar PECAM-1 staining were observed with
foxf1(+/
) lungs at 4 and 6 days after BHT injury (Fig. 8,
D, F, and H). Consistent with this
finding, we observed a diminished number of CD34-positive alveolar
endothelial cells in foxf1(+/
) lungs at 6 days after BHT
lung injury (Fig. 8, I-J). In contrast, large pulmonary
vessels, which do not express Foxf1, displayed normal PECAM-1 staining
in response to BHT injury (Fig. 8, D-E), suggesting
that the decline in PECAM-1 expression was restricted to alveolar
capillaries and arterioles. Moreover, RNase protection assays
demonstrated that, before BHT injury, foxf1(+/
) lungs
displayed a twofold increase in PECAM-1 mRNA compared with wild-type
lungs (Fig. 9). Although BHT injury of foxf1(+/
) lungs caused an 80% reduction in PECAM-1
expression at 6 days, only minor decreases were observed in wild-type
lungs (Fig. 9). Furthermore, PECAM-1 has been implicated in vascular permeability (34) and in protection of endothelial cells
against apoptosis (11, 18), suggesting that
decreased PECAM-1 expression in foxf1(+/
) lungs may
contribute to an increase in BHT-mediated apoptosis of alveolar
endothelial cells (Figs. 5 and 6).

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Fig. 8.
BHT injury of foxf1(+/ ) lungs caused a decrease in
platelet endothelial cell adhesion molecule (PECAM)-1-staining alveolar
endothelial cells. At various intervals after BHT lung injury, lung
sections were prepared from either wt (A, C,
E, G, and I) or
foxf1(+/ ) (B, D, F,
H, and J) mice and used for
immunohistochemical staining with either PECAM-1 or CD34
antigen antibodies to visualize endothelial cells. Pulmonary PECAM-1
expression was similar in untreated wt (A) and
foxf1(+/ ) mice (B). Although BHT exposure did
not affect PECAM-1 protein staining in wt lungs (C,
E, G, and I), the levels and
distribution of alveolar PECAM-1 or CD34 staining were significantly
diminished in foxf1(+/ ) lungs (D, F,
H, and K) at 6 days after BHT lung injury. In
contrast, large pulmonary vessels (Ar), which do not express Foxf1,
displayed normal PECAM-1 staining in response to BHT injury.
Magnification: A-F, I, and J are
at ×200; G and H are at ×600.
|
|

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|
Fig. 9.
RNase protection assays identified altered expression of genes
associated with BHT-induced lung hemorrhage phenotype in adult
foxf1(+/ ) mice. At various intervals after BHT treatment,
total lung RNA was prepared from either wild-type or
foxf1(+/ ) mice and used to analyze for Flk-1, PECAM-1,
vascular endothelial cadherin (VE Cad), vascular endothelial growth
factor (VEGF), bone morphogenetic protein-4 (BMP-4), surfactant protein
(SP)-B, heme oxygenase (HO)-1, vimentin, interleukin (IL)-6, and Cyclo
mRNA levels by RNase protection assay. Each individual sample was
normalized to its corresponding cyclophilin levels as described in
MATERIALS AND METHODS. Numbers represent averages of mRNA
levels for each of the different time points after BHT treatment with
respect to untreated wt lungs. Note that expression of Flk-1, PECAM-1,
VE Cadherin, and BMP-4 is elevated in untreated foxf1(+/ )
lungs (day 0).
|
|
BHT-induced pulmonary hemorrhage of
foxf1(+/
) mice is associated with
diminished pulmonary expression of Flk-1, BMP-4, VE cadherin, and SP-B.
To identify genes whose altered expression was associated with
lung hemorrhage after BHT injury, RNase protection assays were performed in duplicate. Examination of Flk-1, BMP-4, PECAM-1, and VE
cadherin mRNA levels in adult foxf1(+/
) and wild-type lungs before BHT injury demonstrated that their expression was increased in heterozygous lungs (Fig. 9). Because these proteins are
critical for pulmonary mesenchymal cell homeostasis and function (5, 11, 12, 18, 27, 34), their compensatory increased expression may play a role in survival of foxf1(+/
) mice.
Furthermore, we found that VEGF expression was not significantly
affected in either wild-type or foxf1(+/
) mice after BHT
injury, but an 18-fold decline in its receptor Flk-1 mRNA was observed
by day 6 in foxf1(+/
) lungs (Fig. 9). Because
Flk-1 mediates endothelial cell proliferation and survival
(5), its reduced expression in foxf1(+/
)
lungs after BHT injury correlates with a diminished number of
PECAM-1-positive endothelial cells. Interestingly, BHT injury of
wild-type lungs caused increased expression of BMP-4, which is
consistent with their roles in mediating lung branching morphogenesis
and proliferation of mesenchymal cells that may be involved in the
repair process (4, 45). In contrast, expression of BMP-4
mRNA was decreased in foxf1(+/
) lungs at day 6 after BHT injury, and their reduced levels are associated with
defective lung injury repair. Furthermore, foxf1(+/
) lungs
displayed a sudden reduction in VE cadherin and SP-B mRNA levels at 6 days after BHT injury (Fig. 9), a finding that may explain
increased permeability in the foxf1(+/
) lungs (12,
40). The foxf1(+/
) lungs also exhibited a drastic
increase in IL-6 at day 6 after BHT injury, perhaps
consistent with the role of IL-6 in protection from hyperoxic lung
injury (44). Interestingly, wild-type and
foxf1(+/
) lungs displayed similar induced expression of
heme oxygenase-1 (Fig. 9), an enzyme that is implicated in protection
of endothelial cells against acute lung injury (36, 46).
Finally, we did not observe significant changes in mRNA levels of
vimentin (Fig. 9) and lung Kruppel-like factor (data not shown), the
latter of which is a transcription factor required for normal lung
development (43).
 |
DISCUSSION |
In a previous study, we reported that ~55% of newborn
foxf1(+/
) mice died of severe lung hemorrhage and that the
severity of their pulmonary abnormalities correlated with an 80%
reduction in Foxf1 mRNA levels [designated as low
foxf1(+/
); see Ref. 21]. Defects in
formation of lung alveoli and capillaries were observed in low
foxf1(+/
) newborn mice, suggesting that wild-type levels of Foxf1 are required for normal lung development (21).
The pulmonary hemorrhage was associated with reduced expression of SP-B, VEGF receptor 2 (Flk-1), BMP-4, and lung Kruppel-like factor and
T-box transcription factors, which are critical for lung morphogenesis and function. Interestingly, wild-type pulmonary levels of these regulatory genes and Foxf1 mRNA were observed in a subset of newborn foxf1(+/
) mice that did not exhibit lung hemorrhage
[designated as high foxf1(+/
) mice]. Although high
foxf1(+/
) mice had normal life spans and their lungs
appear morphologically normal, the fact that BHT injury was sufficient
to induce a lethal pulmonary hemorrhage suggests that
foxf1(+/
) lungs had limited capacity to respond to
cellular damage. Consistent with a critical role of wild-type Foxf1
levels for lung morphogenesis (21), BHT-induced foxf1(+/
) lung hemorrhage was associated with a 10-fold
decline in pulmonary Foxf1 mRNA without compensatory increases in
expression of a related family member, Foxf2. The BHT-induced decrease
in pulmonary Foxf1 levels in foxf1(+/
) mice was associated
with significant decreases in expression of Flk-1, PECAM-1, VE
cadherin, BMP-4, SP-B, and bcl-w genes, all of which are critical for
lung morphogenesis and alveolar endothelial cell survival required for
normal lung repair. These results suggest that Foxf1 plays an important
role in regulating the transcriptional network of genes involved in
mediating lung morphogenesis and repair in response to pulmonary injury.
We used TEM to confirm that BHT-injured foxf1(+/
)
lungs possessed apoptotic alveolar endothelial cells and had
damage to capillaries, both of which contribute to the severe pulmonary hemorrhage. We found that BHT injury of foxf1(+/
) lungs
caused alveolar endothelial cells to express undetectable levels of
PECAM-1 protein, which is required for protection against
apoptosis (11, 18), and increased vascular
permeability (34). Our results suggest that diminished
PECAM-1 expression may contribute to elevated apoptosis of
alveolar endothelial cells in BHT-injured foxf1(+/
) lungs.
Interestingly, we found that BHT lung injury did not cause apoptosis of Foxf1-expressing bronchiolar smooth muscle cells, suggesting that apoptosis of this cell type does not contribute to pulmonary hemorrhage. This result is in contrast to the newborn low
foxf1(+/
) mice, which displayed increased
apoptosis of the smooth muscle cells and disruption of its
interface with the bronchiolar epithelial cell layer (21).
This discrepancy in damage of the smooth muscle cell layer of
foxf1(+/
) lungs may reflect the fact that BHT injury is
restricted to the pulmonary epithelial and endothelial cells. We also
found that PCNA staining and expression of the proliferation-specific
FoxM1B gene remained elevated in foxf1(+/
) lungs at 6 days
after BHT injury, suggesting that sustained proliferation occurs in
response to an increase in alveolar apoptosis. Together, our
data suggest that elevated apoptosis of alveolar endothelial
cells in BHT-injured foxf1(+/
) lungs causes increased capillary permeability, contributing to a severe lung hemorrhage phenotype.
Interestingly, adult foxf1(+/
) lungs express wild-type
levels of Foxf1 mRNA and exhibit increased expression of Flk-1, BMP-4, PECAM-1, and VE cadherin. Because these proteins are critical for
pulmonary mesenchymal cell homeostasis and function (4, 5, 11,
12, 18, 34, 45), their elevated expression may compensate for
initial defects in pulmonary septation and may contribute to survival
of Foxf1 heterozygous mice. BHT-induced lung hemorrhage of adult
foxf1(+/
) mice was associated with a drastic reduction in
expression of the Flk-1, PECAM-1, and VE cadherin genes, whereas the
expression of these genes was only slightly diminished in wild-type
lungs after BHT injury. Furthermore, their large reduction in
foxf1(+/
) lungs at 6 days after BHT injury (Fig. 9) cannot
be solely explained by the loss of endothelial cells because the
injured foxf1(+/
) lungs exhibited only a 50% reduction in
-Gal-positive alveolar endothelial cells (Fig. 3E). Interestingly, potential Foxf1-binding sites (38) were
found in distinct enhancer regions that are required for correct
endothelial expression of the mouse Flk-1 (CATTGTTTATGgA) and VE
cadherin (
635 bp, CAGTATTTGTAAA) promoters in transgenic mice
(14, 23) as well as in the human PECAM-1 promoter (
48
bp, GAGTGTTTACTCt) region (3). The finding that potential
Foxf1-binding sites are found in the functional Flk-1, VE cadherin, and
PECAM-1 promoter regions suggests that Foxf1 may be directly regulating
their expression. These proteins are also known to be involved in
endothelial cell survival, vascular morphogenesis, and maintenance of
capillary integrity (5, 11, 12, 15, 18, 34), suggesting
that their decreased expression is likely contributing to lung
hemorrhage. Recent studies have implicated endothelial cells as
critical for the morphogenesis of liver and pancreas, suggesting that
they secrete growth factors or provide cell-cell contacts essential for
organ development (26, 32). It is tempting to speculate that the reduction in alveolar endothelial cells after BHT injury of
foxf1(+/
) lungs may also contribute to the loss of growth factors critical for lung morphogenesis and repair in response to
cellular damage.
We also found that BHT injury of wild-type lungs caused increased
levels of BMP-4, which is consistent with their roles in mediating lung
development (4, 45), and their increased expression is
recapitulated during cellular repair in response to lung injury. In
contrast, BHT-injured foxf1(+/
) lungs displayed an
opposite response with decreased pulmonary expression of BMP-4 mRNA,
which was associated with defective lung injury repair and subsequent development of pulmonary hemorrhage. Our current BHT injury studies and
previous studies in newborn foxf1(+/
) mice
(21) demonstrate that diminished pulmonary Foxf1 levels
correlate with reduced expression of BMP-4. The finding that the BMP-4
promoter contains binding sites for Foxf1 (38) and
significant reduction in mesodermal expression of BMP-4 was found in
foxf1(
/
) embryos provides further support for this
concept (29). Moreover, although expression of SP-B was
elevated during the first 4 days after BHT injury, its mRNA levels
decreased in foxf1(+/
) mice at day 6,
coinciding with lung hemorrhage (Fig. 9). Exposure of
SP-B(+/
) mice to hyperoxia caused increased severity of
pulmonary edema and hemorrhage (40), indicating that
diminished SP-B levels compromise its protective role in the lung.
These findings are consistent with the concept that wild-type levels of
Foxf1 are required to regulate genes necessary for pulmonary
morphogenesis and endothelial cell homeostasis during cellular
proliferation in response to lung injury.
In summary, we demonstrated that adult foxf1(+/
) mice were
highly susceptible to BHT-induced lung injury. BHT injury caused lethal
pulmonary hemorrhage in adult foxf1(+/
) mice and was
associated with a 10-fold decrease in pulmonary Foxf1 expression and an
increase in apoptosis of alveolar endothelial cells.
Furthermore, BHT injury of foxf1(+/
) lungs caused an
aberrant reduction in levels of Flk-1, PECAM-1, VE cadherin, BMP-4,
SP-B, and bcl-w mRNA, which are critical for cell survival and lung
morphogenesis during repair after pulmonary injury. Collectively, our
data suggest that Foxf1 is a potential target for therapeutic
intervention during a variety of pulmonary diseases involving defective
lung repair.
 |
ACKNOWLEDGEMENTS |
We thank Fengli Guo, Ana Bursick, Mara Sullivan, and Jean Clark for
excellent technical assistance and Pradip Raychaudhuri for critically
reviewing the manuscript.
 |
FOOTNOTES |
This work was supported by National Institutes of Health Grants
HL-62446 (R. H. Costa), HL-56387 (J. A. Whitsett), HL-41496 (J. A. Whitsett), and CA-76541 (D. B. Stolz).
Address for reprint requests and other correspondence:
R. H. Costa, Dept. of Molecular Genetics (M/C 669), Univ. of
Illinois at Chicago, College of Medicine, 900 S. Ashland Ave., Rm. 2220 MBRB, Chicago, IL 60607-7170 (E-mail:
Robcosta{at}uic.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 11, 2002;10.1152/ajplung.00463.2001
Received 14 December 2001; accepted in final form 10
January 2002.
 |
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