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Regulation of embryonic lung vascular development by vascular endothelial growth factor receptors Flk-1 and Flt-1.

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Growth & Development
THE ANATOMICAL RECORD 290:958–973 (2007)
Regulation of Embryonic Lung
Vascular Development by Vascular
Endothelial Growth Factor
Receptors, Flk-1 and Flt-1
YASUTOSHI YAMAMOTO,1,2 ISAO SHIRAISHI,2 PING DAI,1
KENJI HAMAOKA,2 AND TETSURO TAKAMATSU1*
1
Department of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine
Graduate School of Medical Science, Kyoto, Japan
2
Department of Pediatric Cardiology and Nephrology, Kyoto Prefectural University of
Medicine Graduate School of Medical Science, Kyoto, Japan
ABSTRACT
The biological effects of vascular endothelial growth factor A (VEGFA) are mediated by fetal liver kinase-1 (Flk-1) and fms-like tyrosine kinase-1 (Flt-1). In lung tissue, VEGF-A is diffusely expressed throughout
the embryonic stages, whereas the development of vascular endothelial
cells is not uniform. Noting the signaling properties of the two receptors,
we hypothesized that Flk-1 and Flt-1 regulate the embryonic development
of lung vasculature. We herein show the spatiotemporal expression and
experimental inhibition of Flk-1 and Flt-1 of embryonic mouse lung tissue. When Flk-1 was predominantly expressed (embryonic day [E] 9.5–
E13.5), then vascular endothelial cells actively proliferated. When Flt-1
was enhanced (E14.5–E16.5), these cells less actively proliferated,
thereby constituting organized networks. The treatment of cultured lung
buds (E11.5) with antisense oligonucleotides complementary to Flk-1
inhibited branching of capillaries and proliferation of endothelial cells. In
contrast, the inhibition of Flt-1 promoted the branching of capillaries and
enhanced proliferation of endothelial cells. Of interest, inhibition of Flt-1
promoted Flk-1 expression. These results suggest that the two VEGF-A
receptors regulate pulmonary vascular development by modulating the
VEGF-A signaling. Anat Rec, 290:958–973, 2007. Ó 2007 Wiley-Liss, Inc.
Key words: vascular endothelial growth factor receptors
(VEGFRs); Flk-1; Flt-1; angiogenesis; vasculogenesis; lung; mouse; embryo
Mouse lung development starts at embryonic day (E)
9.5, when the endodermal foregut evaginates into the surrounding splanchnic mesoderm. Subsequently, the two
lung buds elongate and repeat branching in a highly reproducible manner, thus giving rise to the bronchial tree
(Ten Have-Opbroek, 1991; Hogan, 1999). Fibroblast
growth factor 10 (FGF10) and bone morphogenetic protein 4 (BMP4) and the regulation of these molecules at
the transcription level by the sonic hedgehog (shh),
patched (ptc), smoothened (smo), and Gli pathways have
been clarified (Warburton et al., 2000). On the other
hand, abundant mesodermal mesenchymal cells, which
surround the developing bronchial tree, differentiate into
Ó 2007 WILEY-LISS, INC.
This article contains supplementary material available via
the internet at http://www.interscience.wiley.com/jpages/19328486/suppmat.
Grant sponsor: Japan Society for the Promotion of Science.
*Correspondence to: Tetsuro Takamatsu, Department of Pathology and Cell Regulation, Kyoto Prefectural University of
Medicine Graduate School of Medical Science, KawaramachiHirokoji, Kamigyo, Kyoto, Japan 602-8566.
E-mail: ttakam@koto.kpu-m.ac.jp
Received 3 May 2006; Accepted 14 May 2007
DOI 10.1002/ar.20564
Published online in Wiley InterScience (www.interscience.wiley.
com).
VEGF RECEPTORS DURING LUNG DEVELOPMENT
vascular endothelial cells in alignment but they are significantly distant from the developing bronchial airway
(Gebb and Shannon, 2000; Schachtner et al., 2000), thus
eventually forming lung vasculature. Although the lung
vasculature and the bronchial tree develop simultaneously during embryogenesis, very little is known regarding the development of the lung vasculature (Stenmark
and Gebb, 2003).
Vascular endothelial growth factor-A (VEGF-A) plays a
critical role in vasculogenesis and angiogenesis by regulating vascular endothelial cell proliferation and differentiation (Carmeliet et al., 1996; Ferrara et al., 1996).
VEGF-A also plays an important role during the pulmonary vascular development, and its splice variants are
strictly regulated (Ng et al., 2001). The biological effects
of VEGF-A are mediated by two receptor tyrosine
kinases, fms-like tyrosine kinase-1 (Flt-1) and fetal liver
kinase-1 (Flk-1; Shibuya et al., 1990; de Vries et al., 1992;
Terman et al., 1992). These differ considerably regarding
their signaling properties, such as the affinity for VEGFA and kinase activity (Waltenberger et al., 1994; Seetharam et al., 1995; Sawano et al., 1996). Flk-1 null
mutants lack mature endothelial and hematopoietic cells
(Shalaby et al., 1995). In contrast, mice lacking Flt-1
demonstrate a disorganized vasculature and an increased
number of endothelial progenitor cells (Fong et al., 1995).
These findings indicate that Flk-1 positively regulates
the VEGF-A signals, whereas Flt-1 negatively regulates
the VEGF-A signals (Park et al., 1994).
Although VEGF-A is predominantly expressed by epithelial cells in the lung (Acarregui et al., 1999; Gebb and
Shannon, 2000; Ng et al., 2001), vascular endothelial
cells emerge and differentiate in areas significantly distant from the developing bronchial airway and acini
(Schachtner et al., 2000). Furthermore, Flk-1–positive
cells maintain a distinct spatial relationship relative to
the branching epithelium and they are only present
within a characteristic proximity of the epithelium in the
pseudoglandular stage (within approximately three to six
cell diameters; Gebb and Shannon, 2000). Factor(s) other
than VEGF-A itself might be involved in regulating vascular endothelial cell proliferation and differentiation.
Because Flk-1 and Flt-1 transduce the VEGF-A signals in
different ways, we hypothesized that the coordinated
expression of Flk-1 and Flt-1 may thus be an essential
process in the constitution of the highly organized lung
capillary networks.
By means of morphometry with confocal microscopy,
mRNA quantification by real-time polymerase chain reaction(PCR), 5-bromo-20 -deoxyuridine (BrdU) incorporation
into developing vascular endothelial cells, and target inhibition of mRNA or proteins, we herein demonstrate
that Flk-1 and Flt-1 regulate vascular endothelial cell
proliferation, differentiation, and the subsequent development of the pulmonary vasculature. When Flk-1 expression is enhanced, the vascular endothelial cells actively
proliferated and differentiated. When Flt-1 was dominant, vascular endothelial cells less actively proliferated
and differentiated. The treatment of cultured lung buds
(E11.5) with antisense oligodeoxynucleotides (AS-ODNs)
complementary to Flk-1 thus resulted in an insufficient
branching of the capillaries, and an impaired proliferation, while also promoting the apoptosis of endothelial
cells and decreased the ephrin-B2–positive arterial cell
lineage. In contrast, the inhibition of Flt-1 with AS-ODNs
959
promoted the branching of the capillaries, an enhanced
proliferation of endothelial cells, and an increased
ephrin-B2–positive arterial cell lineage. Of interest, the
inhibition of Flt-1 in the lung buds promoted Flk-1
expression; however, the inhibition of Flk-1 did not
enhance Flt-1 expression. These results provide the first
demonstration that the differential expression of VEGF-A
receptors Flk-1 and Flt-1 control the development of the
pulmonary vasculature.
MATERIALS AND METHODS
Animal Preparation
Lungs were taken from the embryos (E9.5–E18.5) of
pregnant ICR mice. Fetal lung buds taken at E11.5 from
pregnant ICR mice were obtained for an in vitro functional study using either AS-ODNs or specific antibodies
to Flk-1 and Flt-1. All animal care and all experimental
procedures were approved by the Committee for Animal
Research, Kyoto Prefectural University of Medicine.
Immunohistochemistry and F-Actin Staining
Whole-mount lung immunofluorescence studies using
an anti–platelet endothelial cell adhesion molecule-1 (anti–
PECAM-1) antibody and subsequent sectioning were performed as follows. To elucidate the spatial relationship
between vascular endothelial cells and pulmonary epithelial cells, F-actin staining along with PECAM-1 staining
was performed: lungs were fixed with 4.0% paraformaldehyde (PFA), and permeabilized with 0.2% Triton X-100 in
phosphate-buffered saline (PBS). For F-actin staining,
lung tissues were treated with Texas Red-X phalloidin
(1:50; Molecular Probes). Thereafter, the tissue specimens
were incubated with anti–PECAM-1 antibody (1:200;
Pharmingen). After being rinsed, the tissue specimens
were incubated with Alexa 488–conjugated anti-rat IgG
(1:200; Molecular Probes). Next, tissue specimens were
cover-slipped using 50% glycerin with 10-mg/ml 1,4-diazabicyclo(2,2,2)octane (DABCO). Immunohistochemistry
for VEGF, Flk-1, Flt-1, PECAM-1, and ephrin-B2 was
performed as follows: The lungs were taken from the
embryos and fixed with 4% PFA. They were then embedded in OCT compound. Sections on glass slides were dried
and fixed with 4% PFA and then were treated with 100%
ethanol. Next, the slides were incubated with primary
antibodies. The following antibodies were used: rat anti–
PECAM-1 (1:500; Pharmingen), rabbit anti-VEGF (1:500;
Santa Cruz), rat anti–Flk-1 (1:1,000; kindly donated by
Dr. Shin-Ichi Nishikawa, Center for Developmental Biology, Kobe, Japan), rat anti–Flt-1 (1:500; kindly donated
by ImClone system), rabbit anti-ephrin–B2 (1:500; Santa
Cruz), and rabbit anti-pro surfactant protein C (pro SP-C;
1:1,000; CHEMICON).
Confocal Laser Scanning Microscopy
The stained specimens were observed by a confocal
laser scanning microscope (CLSM: Olympus FLUOVIEW
FVX system; Olympus, Tokyo, Japan) equipped with an
oil immersion objective (Plan Apo 360, NA 5 1.4; Olympus) and a dry objective (Uplan Fl 310, NA 5 0.3; Olympus). An argon-krypton laser produced excitation bands
at 488 nm for Alexa 488, bands at 568 nm for Alexa 594,
and monochromatic light for differential interference contrast (DIC) images. Fluorescent images were collected
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YAMAMOTO ET AL.
with emission filters for 510–550 nm for Alexa 488 and
585–610 nm for Alexa 594. Simultaneous images (800 3
600 pixels, 12 bits each) of either Alexa 488- or 594-labeled and DIC images were acquired and stored. Serial
optical sections (up to 30 sections) were obtained by moving the sample stage at intervals of 1.0 mm. Stereo pair
images were generated by reconstructing the series of optical sections using the CLSM software package.
Morphometry
The numbers of PECAM-1–, Flk-1–, Flt-1–, ephrin-B2–,
and pro SP-C–positive cells in each high power field
(objective lens, 360) were counted using a computerized
image analysis system (NIH image). The percentages of
PECAM-1–, Flk-1–, or Flt-1–positive cells in total cells
were calculated. The percentage of pro SP-C–positive
cells out of the total pulmonary epithelial cells was calculated. The ratio of arterial vascular endothelial cells was
defined as the percentage of ephrin-B2–positive and
PECAM-1–positive cells in all PECAM-1–positive cells.
VEGF-positive area for the same field (21,590 square pixels) was measured and expressed as mean pixels. For
each lung bud, the average number of microvessel branch
points was calculated essentially as previous described
with minor modifications (Ruhrberg et al., 2002) and
determined by stereogram in 3 randomly chosen areas of
the peripheral lung buds. The data were obtained from
three to five separate experiments.
Quantitative Real-Time PCR
Only the peripheral regions of the lung tissues were
dissected under a stereomicroscope and then they were
harvested to isolate RNA. Quantitative real-time PCRbased measurements of RNA abundance were carried out
using gene-specific double fluorescent probes (designed
by ABI) and real-time PCR instruments (ABI, PRISM
7700). The amounts of PECAM-1, Flk-1, Flt-1, and ephrin-B2 mRNA were standardized by 18S ribosomal RNA.
The quantitative data are the average of two independent
experiments. Each mRNA-quantification was done with
the total RNA isolated from 10 to 15 embryos.
In Situ Hybridization
Total RNA was extracted from E14.5 fetal mouse lung
tissue. A 419-bp VEGF cDNA fragment, encoding exon 1
to 5, was generated by the reverse transcriptase-PCR
(RT-PCR). The primer pair was designed as follows: forward primer, 50 -GGGGATCCATGAACTTTCTGCTGTC
TTGGGTGCACTGG-30 ; reverse primer, 50 -AAAAGCTTC
TGGCTTTGTCCTGTCTTTCTTTGGTCTGC-30 (Breier et al.,
1992). The PCR products were subcloned into pGEM-3Zf
(6) Vector (Promega) and linealized. Then both sense and
antisense RNA probes were prepared by in vitro transcription using a DIG RNA Labeling Kit (SP6/T7; Boehringer Mannheim, Germany). Six-micrometer frozen
sections of E14.5 embryonic lung were fixed with 4% PFA
for 5 min, treated with 1.0 mg/ml proteinases K for
15 min, acetylated for 10 min, and then were dehydrated
with graded ethanol. After the pretreatment, the sections
were hybridized with 1 mg/ml VEGF RNA probe for 20 hr
at 558C. Signals were detected using a DIG Nucleic Acid
Detection Kit (Boehringer Mannheim, Germany).
Lung Bud Culture
The serum-free lung bud culture was done essentially
as previously described with minor modifications
(Ohmichi et al., 1998). Fetal lung buds were taken from
E11.5 ICR pregnant mice (treated group; n 5 10–15, control group; n 5 10–15) and then were placed in culture
plates with 12 separate wells containing 600 ml of medium (Dulbecco’s modified Eagle’s medium containing
0.38% [w/v] NaHCO3, 100 units/ml penicillin, 100 mg/ml
streptomycin). All the explants were incubated in a
humidified chamber with 95% air and 5% CO2. The
explants were photographed after 2, 24, 48, and 96 hr
and then were fixed with 4% PFA. F-actin and PECAM-1
were detected by immunohistochemistry as previously
described. Images were taken using a CLSM (Olympus).
Each experiment was repeated at least three times.
AS-ODNs Targeted to Flk-1, Flt-1
The inhibition of Flk-1 or Flt-1 during lung development in culture was examined using AS-ODNs (1.0 mM
and 2.5 mM, respectively; Proligo, Japan) as described in
a previous report with some modifications (Ohmichi
et al., 1998). The sequences are designed to inactivate the
initiation codon of the published mouse Flk-1 or Flt-1
mRNA as follows: 50 -AGGUGCAGGAUGGAGAGCAA-30
for Flk-1 (NCBI GenBank X59397), 50 -UUGCUCAC
CAUGGUCAGC-30 for Flt-1 (NCBI GenBank D88689).
Scrambled sequence oligodeoxynucleotides (S-ODNs)
were used as a control. After 48 hr, the explants were
fixed and double stained with Texas Red-X–conjugated
phalloidin and antibody against PECAM-1 as described.
The development of lung vascular endothelial cells was
evaluated by using the CLSM. To verify the uptake of
oligodeoxynucleotides into endothelial cells, fluorescein
isothiocyanate–labeled S-ODNs were used in the same
manner. A specific decrease in the gene copy was confirmed by RT-PCR.
RT-PCR
RT-PCR was done according to the manufacturer’s recommendations (Roche). Total RNA was extracted from
lung explants using RNeasy Mini Procedure kit (QIAGEN).
Short- and single-strand cDNA was prepared from total
RNA using SuperScriptTMII RNase H-Reverse Transcriptase (Invitrogen). The following sets of primers were
designed (Choi et al., 1998; Akeson et al., 2000). b-actin:
forward primer, 50 -TGGAATCCTGTGGCATCCATGAA
AC-30 ; reverse primer, 50 -TAAAACGCAGCTCAGTAAC
AGTCCG-30 . Flk-1: forward primer, 50 -CACCTGGCAC
TCTCCACCTTC-30 ; reverse primer, 50 -GATTTCATCCCA
CTACCGAAA-30 . Flt-1: forward primer, 50 -CTCTGATGG
TGATCGTGG-30 ; reverse primer, 50 -CATGCGTCTGGCC
ACTTG-30 . PCR conditions were as follows: denaturation
at 948C for 30 sec, followed by annealing at 558C for
60 sec, and extension at 728C for 60 sec.
Inhibition of Flk-1 or Flt-1 by Antibodies
Angiogenesis inhibitors (DC101; rat anti-mouse Flk-1
antibody, MF-1; goat anti-mouse Flt-1 antibody, kindly
VEGF RECEPTORS DURING LUNG DEVELOPMENT
gifted from ImClone Systems, 50 mg/ml) were added to
the culture medium. After 48 hr, the explants were fixed
and double stained with Texas Red-X–conjugated phalloidin and antibody against PECAM-1 as previously
described. The development of lung vascular endothelial
cells was evaluated by using the CLSM. Each experiment
was repeated at least three times.
Cell Proliferation Assay
Flash labeling with BrdU and the subsequent determination of labeling index was performed as previously
described (Ruhrberg et al., 2002). Timed pregnant mice
(E10.5, E12.5, E14.5, and E18.5) were intraperitoneally
injected with 0.0125 mg/g body weight BrdU (Sigma) in
PBS. One hour after the injection, the embryos were dissected from the uterus and the lungs were fixed with 4%
PFA and embedded into OCT compound. Vascular endothelial cells were stained with anti–PECAM-1 antibody
(1:300; Pharmingen) and then were detected by Alexa
594–conjugated anti-rat IgG (1:300; Molecular Probes).
Next, the sections were incubated with 4 N HCl for 30
min at room temperature, rinsed in PBS once, and then
incubated with anti-BrdU antibody (1:500; Exalpha Biologicals) for 30 min at 378C. Alexa 488–conjugated antisheep IgG (1:1,000; Molecular Probes) was used for the
detection of BrdU. The sections were incubated with 4,6diamidino-2-phenylindole (DAPI) for 30 min at room
temperature. The slides were observed by conventional
fluorescence microscopy (Olympus AX70; Olympus,
Tokyo, Japan), and the images were analyzed using a fluorescence imaging system (IPLab3.5.2). The labeling
index of pulmonary vascular endothelial cells was defined
as a percentage of the BrdU-positive and PECAM-1–positive cells in total PECAM-1–positive cells. The data were
obtained from three separate experiments.
Detection of Apoptosis
Apoptotic cell death was detected by immunofluorescence with antibody against single-stranded DNA
(1:1,000; IBL, Japan; Frankfurt, 2004). The incidence
of apoptosis in vascular endothelial cells was defined as
the percentage of single-stranded DNA-positive and
PECAM-1–positive cells out of all PECAM-1–positive
endothelial cells. The data were obtained from three separate experiments.
Statistical Analysis
All values were presented as the mean 6 SD. Statistical significance between the groups was determined by a
one-way analysis of variance, followed by either the
Fisher or the unpaired t-test, as appropriate. A value of
P < 0.05 was considered to be significant.
RESULTS
Fetal Lung Vascular Development Depends
on the VEGF Receptors Flk-1 and Flt-1
Based on the relationship between vascular endothelial
cells and bronchial epithelial cells and in accordance with
literature (Burri, 1984; deMello et al., 1997), we divided
lung vascular development into the following four stages:
961
stage I, E9.5–E10.5; stage II, E11.5–E13.5; stage III,
E14.5–E16.5; and stage IV, E17.5–E18.5.
Stage I (E9.5–E10.5): A few PECAM-1–positive vascular endothelial cells appeared in the distal mesenchyme
surrounding the developing airway of the lung bud (Fig.
1A,I; 4.3 6 1.0%). Flk-1–positive cells were distributed
surrounding the developing airway of the lung bud. (Fig.
2A,I; 40.5 6 5.6%). Flt-1–positive cells were hardly visible
at this stage in the distal mesenchyme surrounding the
developing airway of the lung bud (Fig. 2E), except in the
pulmonary artery in the proximal portion of the lung bud
(Fig. 2J; 5.7 6 4.5%, Supplementary Fig. 1). There were
abundant BrdU- and PECAM-1–positive cells surrounding the developing airway of the lung bud (Fig. 1J; 67.0 6
10.4%).
Stage II (E11.5–E13.5): PECAM-1–positive cells increased
in number (Fig. 1B,I; 11.6 6 2.6%), and they formed
immature and various-sized vascular lumina (Fig. 1F,
stereograms). Flk-1–positive cells were still abundantly
expressed (Fig. 2B,I; 43.0 6 8.6%). A small number of Flt1–positive cells appeared in the distal mesenchyme
surrounding the developing airway of the lung bud (Fig.
2F,J; 15.9 6 5.6%). Quantification of mRNA by real-time
PCR revealed that PECAM-1, Flk-1, and Flt-1 mRNA
expression was 3.1-fold, 1.6-fold, 2.1-fold higher than that
of stage I, respectively. Abundant BrdU-positive and
PECAM-1–positive cells surrounded the bronchial acini
(Fig. 1J; 62.8 6 16.8%).
Stage III (E14.5–E16.5): PECAM-1–positive cells increased in number and encircled the branching airways
(Fig. 1C,I; 19.5 6 1.2%). They ultimately constituted
organized capillary networks with uniform-sized lumina
(Fig. 1C,G; stereograms). As has been previously reported
(Acarregui et al., 1999), in situ hybridization revealed
that VEGF mRNA was primarily expressed in bronchial
epithelial cells and, to a lesser extent, in mesenchymal
cells (Supplementary Fig. 2). Flk-1–positive cells were
still abundantly distributed (Fig. 2C,I; 47.0 6 12.8%). Flt1–positive cells (arrowheads in Fig. 1S) were increased in
number and surrounded the branching airways (Fig.
2G,J; 31.9 6 10.9%). The quantification of mRNA revealed
PECAM-1, Flk-1, and Flt-1 mRNA expression to be 5.1fold, 1.9-fold, and 2.3-fold higher than that of stage I,
respectively. The ratio for BrdU-positive vascular endothelial cells showed a significant decrease in comparison
to that for stages I and II (Fig. 1J; 43.5 6 16.0%).
Stage IV (E17.5–E18.5): PECAM-1–positive vascular
endothelial cells were located closer to alveolar epithelial
cells (Fig. 1D) with more uniform-sized lumina (Fig. 1H;
stereograms). Although the number of PECAM-1–positive
cells increased (Fig. 1D,I; 41.0 6 4.8%), the number of
Flk-1–positive cells (Fig. 2D,I; 26.7 6 4.7%) and Flt-1–
positive cells (Fig. 2H,J; 7.7 6 1.4%) decreased. In addition, the Flk-1 mRNA expression was found to have
decreased in comparison to that of stage III (0.68-fold).
The BrdU incorporation into vascular endothelial
cells significantly decreased in comparison to that for
stages I and II (Fig. 1J; 39.8 6 5.6%). A real-time PCR
analysis of spliced and soluble form of Flt-1, which
also plays a significant role as a decoy for Flk-1–
VEGF signaling (Kendall and Thomas, 1993), showed a
robust up-regulation of soluble Flt-1 at stage IV in
comparison to the former stages (0.77-, 1.03-, and a 4.65fold increase of stage I at stages II, III, and IV, respectively).
962
YAMAMOTO ET AL.
Fig. 1. Four developmental stages of the fetal lung vasculature
based on the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1). A–D: The microscopic appearance of fetal lungs
were divided into the following 4 stages: stage I (A, embryonic day [E]
10.5), stage II (B, E12.5), stage III (C, E14.5), and stage IV (D, E18.5).
A,B,J: Vascular endothelial cells actively proliferated and differentiated
at stage I and II. C,D,J: Vascular endothelial cells less actively prolifer-
ated and differentiated after stage III. A–D: The whole-mount observation of embryonic lung immunostained with PECAM-1 antibody (green)
and F-actin (red). E–H: Stereograms of PECAM-1–positive vascular
endothelial cells. I,J: The quantification of percentage of PECAM-1 (1)
cells (I) and percentage bromodeoxyuridine (BrdU, 1) PECAM-1 (1)
cells (J). *P < 0.05, **P < 0.01. Scale bars 5 50 mm in D (applies to
A–D), 20 mm in H (applies to E–H).
These findings indicate that the morphological transition from loose capillary networks (stages I–II)
into highly organized ones (stage III) is closely associated with the branch formation of bronchial epithe-
lial cells, the spatiotemporal expression of Flk-1 and
Flt-1, and the altering proliferation of vascular endothelial cells. It is also suggested that vascular endothelial cell proliferation is promoted by the predomi-
Fig. 2. Spatiotemporal expression of Flk-1 and Flt-1. There were
abundant Flk-1 (1) cells at stage I–III, and their number decreased at
stage IV. Flt-1–positive cells increased in number at stage III and
decreased at stage IV. A–H: Differential interference microscopy image
and immunofluorescence image of Flk-1 (A–D), and Flt-1 (E–H). I,J:
Quantification of percentage Flk-1 (1) cells (I) and percentage Flt-1
(1) cells (J). **P < 0.01. Scale bars 5 50 mm in D (applies to A–D) and
H (applies to E–H).
964
YAMAMOTO ET AL.
nant expression of Flk-1 (stages I–II), while it is downregulated by the transient expression of Flt-1
(stage III) and by the up-regulation of soluble Flt-1 at
stage IV.
AS-ODNs Targeted to Flk-1 mRNA Inhibit the
Branching of Pulmonary Capillaries While
Impairing the Proliferation of Vascular
Endothelial Cells
To inhibit Flk-1 during the pulmonary vascular development, cultured lung buds at E11.5 (Stage II) were
treated with AS-ODNs complementary to Flk-1 mRNA.
The S-ODNs were used as a control. The down-regulation
in specific gene copy was confirmed by RT-PCR (Fig. 3H).
The treatment of the lung buds with AS-ODN targeted to
Flk-1 decreased the number of PECAM-1–positive vascular endothelial cells and impaired the formation of capillary lumina (compare Fig. 3C with Fig. 3B). The ratio of
PECAM-1–positive cells to the total number of cells was
significantly lower in the Flk-1 AS-ODN–treated buds
(10.5 6 4.4%) than in the S-ODN–treated buds (20.6 6
3.8%; Fig. 3I; P < 0.01). The PECAM-1 mRNA level
decreased in Flk-1 AS-ODN–treated lung buds in comparison to those treated with S-ODN (0.58-fold). Interestingly, the inhibition of Flk-1 also impaired the growth of
bronchial epithelial cells, characterized by large acinus
formation (Fig. 3C). To elucidate the effects of Flk-1 inhibition on the epithelial differentiation, we performed
immunostaining using pro SP-C, which is expressed in alveolar Type II cells. The inhibition of Flk-1 by AS-ODN
attenuated the number of pro SP-C–positive cells in comparison to S-ODN–treated lung buds (Fig. 3L–N; P 5
0.0012). Stereograms of PECAM-1 demonstrated that the
inhibition of Flk-1 impaired the formation of the vascular
branching and capillary lumen (Fig. 3F). The number of
microvessel branch points was significantly smaller in
the Flk-1 AS-ODN–treated lung buds (6.9 6 2.2) than in
the S-ODN–treated ones (21.5 6 6.8; Fig. 3J; P < 0.05).
As shown in Figure 3G, the impaired growth of capillary
lumina (mean diameter, 6.40 6 2.38 mm for anti–Flk-1
antibody-treated buds vs. 15.3 6 4.04 mm for control
buds, P < 0.01) and branching was also confirmed by the
treatment of lung buds with antibody against Flk-1
(DC101). These findings suggest that Flk-1 plays an important role in both the normal growth of the capillary
lumen and the promotion of the pulmonary vascular
branching. To elucidate how the inhibition of Flk-1
impaired the capillary formation and the branching morphogenesis of cultured lung buds, we investigated both
Fig. 3. A–N: Effects of antisense oligodeoxynucleotides (AS-ODNs)
directed to Flk-1 mRNA on fetal lung capillary morphogenesis in culture. The treatment of lung buds with AS-ODN targeted to Flk-1 for 48
hr decreased the number of platelet endothelial cell adhesion molecule-1 (PECAM-1) –positive vascular endothelial cells and impaired the
capillary branching (I and J, compare F with D and E). K: The inhibition
of Flk-1 significantly suppressed the proliferation of vascular endothelial cells. G: The impaired growth of the capillary lumina and branching
was also confirmed by treating the lung buds with antibody against
Flk-1 (DC101). H: Flk-1 AS-ODN decreased the specific gene copy
(reverse transcriptase-polymerase chain reaction). (2), ODN2; S, SODN; AS, AS-ODN. The inhibition of Flk-1 induced a smaller degree of
BrdU incorporation and the detection of apoptotic death
of vascular endothelial cells. The ratio of proliferating
vascular endothelial cells in Flk-1 AS-ODN–treated lung
buds (10.4 6 8.1%) was significantly decreased in comparison to those treated with S-ODN (41.0 6 9.5%; Fig. 3K;
P < 0.01). The incidence of apoptotic death in PECAM-1–
positive vascular endothelial cells was higher in Flk-1
AS-ODN–treated lung buds (5.5 6 4.2%) in comparison to
those treated with S-ODN (2.0 6 0.7%; P < 0.05). These
findings suggest that the impaired growth of the capillary
lumen and vascular branching in Flk-1–inhibited lung
buds was associated with the down-regulated proliferation and enhanced apoptosis of vascular endothelial
cells.
AS-ODNs Targeted to Flt-1 mRNA Inhibit the
Organization of Pulmonary Capillaries During
Fetal Morphogenesis While Promoting the
Proliferation of Vascular Endothelial Cells
To elucidate the role of Flt-1 in fetal pulmonary vascular development, we also inhibited Flt-1 using AS-ODN.
The down-regulation of Flt-1 mRNA was confirmed by
RT-PCR (Fig. 4H). AS-ODN–treated lung buds showed
increased number of PECAM-1–positive vascular endothelial cells (compare Fig. 4C with Fig. 4B). The ratio of
PECAM-1–positive cells to total cells was significantly
higher in Flt-1 AS-ODN–treated buds (40.4 6 11.9%)
than those treated with S-ODN (19.6 6 6.0%; Fig. 4I; P <
0.01). PECAM-1 mRNA expression was also higher in
Flt-1 AS-ODN–treated lung buds than in S-ODN–treated
buds (1.86-fold). Stereograms of lung buds demonstrated
that AS-ODN targeted to Flt-1 impaired the normal formation of the capillary lumen and networks, that is, the
vascular endothelial cells were increased in number, the
capillary lumen was not uniform in size, and the capillary
branching was promoted (Fig. 4F). The number of microvessel branch points significantly increased in the ASODN–treated lung buds (26.3 6 5.1) in comparison to
those treated with S-ODN (17.0 6 4.1; Fig. 4J; P < 0.01).
As shown in Figure 4G, the irregular formation of capillary lumina (mean diameter, 11.4 6 6.2mm for anti–Flt-1
antibody-treated buds vs. 15.3 6 4.0mm for control buds,
p50.16) and the disorganization of capillary networks
were also confirmed by treatment with anti–Flt-1 antibody (MF-1). These findings suggest that Flt-1 plays an
important role in the normal formation of the capillary
lumen, the regulation of capillary branching, and organization of capillary networks. To clarify how AS-ODN targeting to Flt-1 impaired the organization of pulmonary
pro SP-C expression in comparison to the S-ODN–treated lung buds.
N: There are significant reduction in the ratio for pro SP-C–positive
cells / total epithelial cells (%) in the explants treated with AS-Flk-1.
Confocal images of the cultured lung buds without ODN [A, ODN (2)],
treated with S-ODN (B, S-ODN), and treated with AS-ODN targeted to
Flk-1 mRNA (C, AS Flk-1). PECAM-1–positive vascular endothelial
cells are shown in green, F-actin staining in red. D–G: Stereograms of
the cultured lung buds treated without ODN (D), with S-ODN (E), with
AS Flk-1 (F) or with DC101 (G) labeled with anti-PECAM-1 antibody.
*P < 0.05, **P < 0.01. Scale bars 5 50 mm in A–C, 20 mm in D–G,
20 mm in L,M.
VEGF RECEPTORS DURING LUNG DEVELOPMENT
Figure 3.
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YAMAMOTO ET AL.
capillaries, we investigated BrdU incorporation and apoptotic death of vascular endothelial cells. The ratio of proliferating vascular endothelial cells in Flt-1 AS-ODN–
treated lung buds (58.5 6 2.9%) was significantly higher
than those treated with S-ODN (35.4 6 10.6%; Fig. 4K;
P < 0.01). The incidence of apoptotic death in PECAM-1–
positive vascular endothelial cells was lower in the Flt-1
AS-ODN–treated lung buds (1.6 6 1.3%) than in those
treated with S-ODN (3.3 6 0.7%; P < 0.01). These findings suggest that the disorganization of vascular branching produced by Flt-1 AS-ODN is associated with the
up-regulated proliferation of vascular endothelial cells.
Flk-1 Expression Is Enhanced by the
Inhibition of Flt-1
To examine whether the expression of Flk-1 regulates
that of Flt-1 and vice versa, we immunohistochemically
studied Flk-1- or Flt-1 AS-ODN–treated lung buds
stained with Flt-1 and Flk-1 antibodies, respectively.
There were more Flk-1–positive cells in the lung buds
treated with Flt-1 AS-ODN (Fig. 5C) than those treated
with S-ODN (Fig. 5B). The ratio for Flk-1–positive cells to
total cells was significantly higher in the lung buds
treated with Flt-1 AS-ODN (14.2 6 2.2%) than in those
treated with S-ODN (6.5 6 3.3%; P < 0.05, Fig. 5G). Flk-1
mRNA was also increased in lung buds treated with Flt-1
AS-ODN in comparison to those treated with S-ODN (1.3fold). To clarify whether the higher levels of Flk-1 in
explants treated with AS-Flt-1 is just due to increased
number of Flk-1–positive endothelial cells or whether it is
due to increased number of Flk-1 receptors in each endothelial cell, double staining with Flk-1 and PECAM-1 was
performed. Almost all the Flk-1–positive cells also
expressed PECAM-1 (Supplementary Fig. 4A–D), suggesting that the higher expression of Flk-1 was due to the
increased number of Flk-1– and PECAM-1–positive endothelial cells. In contrast, there was no significant difference in the number of Flt-1–positive cells (Fig. 5F,H; P >
0.05) and mRNA (0.99-fold) in the lung buds treated with
Flk-1 AS-ODN in comparison to those treated with SODN.
Inhibition of Flk-1 and Flt-1 Affects the Ephrin-B2
Expression of Vascular Endothelial Cells
To clarify whether the inhibition of Flk-1 or Flt-1 influences the subsequent differentiation of vascular endothelial cells, we investigated the ephrin-B2 expression,
which is exclusively expressed in the arterial vascular endothelial cell lineage. There were few ephrin-B2–positive
cells in the lung buds treated with Flk-1 AS-ODN (Fig.
6B,F,J) in comparison to those treated with S-ODN (Fig.
6A,E,I). The ratio for ephrin-B2–positive and PECAM-1–
Fig. 4. A–K: Effects of antisense oligodeoxynucleotides (AS-ODNs)
directed to Flt-1 mRNA on fetal lung capillary morphogenesis in culture. The treatment of lung buds with AS-ODN targeted to Flt-1 for
48 hr increased the number of PECAM-1–positive vascular endothelial
cells and capillary branching (I and J, compare F with D and E). K:
The inhibition of Flt-1 significantly promoted the proliferation of vascular endothelial cells. G: The irregular formation of the capillary lumina
and the disorganization of capillary networks were also confirmed by
the treatment with anti–Flt-1 antibody (MF-1). H: Flt-1 AS-ODN
positive cells to total PECAM-1–positive cells was significantly smaller in the Flk-1 AS-ODN–treated buds (46.9 6
12.7%) than in S-ODN–treated buds (76.4 6 20.2%; Fig.
6M; P < 0.01). The Ephrin-B2 mRNA level also decreased
more in the Flk-1 AS-ODN–treated lung buds than in the
S-ODN–treated buds (0.53-fold). In contrast, there were
more abundant ephrin-B2–positive cells in Flt-1 ASODN–treated lung buds (Fig. 6D,H,L) than in the SODN–treated buds (Fig. 6C,G,K). The ratio for ephrinB2–positive and PECAM-1–positive cells to the total
number of PECAM-1–positive cells was significantly
higher in the Flt-1 AS-ODN–treated lung buds (94.7 6
6.1%) than in the S-ODN–treated buds (71.5 6 6.0%; Fig.
6N; P < 0.05). The Ephrin-B2 mRNA level slightly
decreased in the Flt-1 AS-ODN–treated lung buds in comparison to those treated with S-ODN (0.81-fold). These
results suggest that the inhibition of Flk-1 down-regulates the arterialization of the embryonic pulmonary vasculature, whereas the inhibition of Flt-1 promotes it.
DISCUSSION
In this study, we present evidence supporting the novel
concept that the spatiotemporal expression of Flk-1 and
Flt-1 regulates the vascular endothelial cell proliferation
and differentiation and subsequent development of the
pulmonary vasculature (summarized in Fig. 7). The inhibition of Flk-1 resulted in reduced branching of capillaries and an impaired proliferation of vascular endothelial
cells. Conversely, the inhibition of Flt-1 promoted vascular branching and enhanced proliferation of vascular
endothelial cells associated with an increased Flk-1
expression. This is the first report to show that the differential expression of the two VEGF receptors, Flk-1 and
Flt-1, is able to regulate embryonic lung vascular development while the emergence of Flt-1 is a key process for
the morphological transition from loose capillary networks into highly organized ones.
Flk-1 and Flt-1 Function on Vascular Formation
According to the previous reports (Gebb and Shannon,
2000; Schachtner et al., 2000) and our immunohistochemical study in vivo, vascular endothelial cells emerge and
differentiate in aligned but they are significantly distant
from the developing bronchial airway and acini. We,
therefore, proposed a novel mechanism of vascular endothelial cell development in addition to the mechanism
due to VEGF gradient.
Two high affinity receptors of VEGF, Flk-1 and Flt-1,
seems to be involved in the fine-tuning mechanisms for
the signal transduction of VEGF because their effects on
vascular endothelial cells considerably differ in their signaling properties. Flk-1 has also been suggested to be a
decreased specific gene copy (reverse transcriptase-polymerase chain
reaction). (2), ODN2; S, S-ODN; AS, AS-ODN. A–C: The confocal
images of the lung buds without treatment [A, ODN (2)], treated with
S-ODN [B, S-ODN]), and treated with AS-ODN targeted to Flt-1 mRNA
(C, AS Flt-1). D–G: Stereograms of the cultured lung buds treated
without ODN (D), with S-ODN (E), with Flt-1 AS-ODN (F), or with MF-1
(G) labeled with anit–PECAM-1 antibody. *P < 0.05, **P < 0.01. Scale
bars 5 50 mm in A–C, 20 mm in D–G.
VEGF RECEPTORS DURING LUNG DEVELOPMENT
Figure 4.
967
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YAMAMOTO ET AL.
major mediator of VEGF, which transduces many downstream effects of VEGF, including proliferation, migration, permeability, and survival signals (Gerber et al.,
1998). In contrast, the function and signaling properties
of Flt-1 appear to be different, depending upon the developmental stages and the cell type (Ferrara et al., 2003).
One important property is that Flt-1 negatively modulates vascular growth by reducing endothelial cell proliferation (Fong et al., 1995, 1999; Kearney et al., 2002).
Our spatiotemporal observations of embryonic lung tissues could be explained by the differential expression and
properties of the two receptors. When Flk-1 was predominantly expressed at stages I–II, vascular endothelial cells
actively proliferated. When the Flt-1 expression was
enhanced at stage III, these cells less actively proliferated
and started to constitute organized vascular networks.
These data indicated that the transition of dominant
receptors from Flk-1 to Flt-1 induces the maturation of
the embryonic lung vasculature. Vascular endothelial
cells thereafter become more mature and better organized
at stage IV, despite a decreased expression of Flt-1. This
phenomenon could be induced by a decreased expression
of Flk-1 at stage IV and also by an increased expression of
spliced variant and soluble form of Flt-1, which has a
decoy effect on the VEGF–Flk-1 signaling as well as the
full-length (membrane-bound) form.
Our data concerning the inhibition of Flk-1 or Flt-1
confirm the signaling properties of the two receptors in
developing lung tissues. The inhibition of Flk-1 induced
both hypocellularlity and less branching of vascular endothelial cells, whereas the inhibition of Flt-1 resulted in
hypercellularity and more branching of endothelial cells.
The inhibition of Flk-1 not only arrests endothelial cell
proliferation and prevents vessel growth, but it also induces the regression of existing vessels by endothelial cell
death (Carmeliet, 2005).
Of interest, the inhibition of Flk-1 also impaired the
growth of bronchial epithelial cells. Our data demonstrated that the inhibition of Flk-1 induced a smaller
degree of pro SP-C expression in comparison to S-ODN–
treated lung buds. A recent report revealed that the Flk-1
inhibition by DC101 disrupted the postnatal alveolar development (McGrath-Morrow et al., 2005), and the treatment of newborn rats with the VEGF receptor inhibitor
SU5416 impaired alveolar morphogenesis (Le Cras et al.,
2002). These findings support the idea that VEGF-A signaling through Flk-1 is a critical facilitator of the crosstalk between epithelial and endothelial cells and the
resultant alveolar development (Del Moral et al., 2006).
Recently, DeLisser et al. (2006) demonstrated that loss of
PECAM-1 function resulted in the disruption of both
proximal and distal lung development without reducing
endothelial cell content. These observations support the
idea that vascular endothelial cell lineage directly signal
back by means of paracrine growth, survival, or morphogenetic factors to influence epithelial development as
seen in the liver development (Matsumoto et al., 2001). In
contrast, the inhibition of Flt-1 did not impair pulmonary
epithelial development, although it did induce both the
hypercellular and disorganized capillary networks. Our
data also demonstrated that the inhibition of Flt-1 did
not influenced pro SP-C expression (Supplementary Fig.
5). This finding suggests that the up-regulation of the
VEGF signals by means of inhibition of Flt-1does not
influence the epithelial cell differentiation, even though
down-regulation has a significant effect in our organ culture models. The transgenic overexpression of VEGF-A in
lung epithelium has been reported to result in abnormal
lung development with dilated epithelial tubes and
increased peritubular vascularity (Zeng et al., 1998). The
overexpression of VEGF164 in distal lung epithelium
caused decreased peripheral capillary network formation
and decreased distal airspace branching (Akeson et al.,
2003). These diverse effects of VEGF on the pulmonary
epithelial and vascular endothelial cells thus suggest that
the spatiotemporal expression of VEGF and downstream
signaling molecules are important not only for the capillary network formation but also for the accompanying
bronchial growth and alveolization. A further investigation is, therefore, needed to elucidate the interactions
between vascular endothelial cells and bronchial epithelial cells during the development of the pulmonary alveolar system.
Negative Regulation of Flk-1
Expression by Flt-1
Although Flk-1 and Flt-1 transduce divergent signal
properties of VEGF, the mechanisms that regulate the
expression of Flk-1 and Flt-1 genes are still not fully
understood. VEGF itself is able to up-regulate both Flk-1
and Flt-1 genes (Barleon et al., 1997; Shen et al., 1998;
Wang et al., 2000). Until recently, it remains uncertain
whether or not a crosstalk mechanism exists between
these two divergent receptors, Flk-1 and Flt-1. It has
recently been shown that the vascular overgrowth seen in
the absence of Flt-1 can be modulated by preventing tyrosine phosphorylation and downstream signaling, thus
suggesting that Flt-1 negatively modulates the blood vessel formation by down-regulating the signaling through
Flk-1 (Roberts et al., 2004). A recent report also demonstrated that placental growth factor binds to Flt-1, thus
resulting in the intermolecular phosphorylation of Flk-1,
and amplifying VEGF-A–driven angiogenesis through
Flk-1 (Autiero et al., 2003). According to our results, the
up-regulation of Flk-1 in response to the inhibition of Flt1 indicates that Flk-1 is at least in part regulated by Flt1. The result that the inhibition of Flk-1 did not affect the
Flt-1 expression raises the possibility that Flt-1 is regulated by an independent mechanism from Flk-1. Therefore, our data raise the fascinating possibility that Flk-1
and Flt-1, and not merely VEGF-A itself, are significantly
involved in the regulation of the development and organization of pulmonary vascular endothelial cells.
VEGF Receptors and Ephrin-B2 Expression
The Eph–ephrin system has been shown to be involved
in the demarcation of arterial and venous boundaries
(Wang et al., 1998). Ephrin-B2 is known to be expressed
in arterial endothelial cells, pericytes, and smooth muscle
cells, whereas EphB4, a receptor for ephrin-B2, is known
to be expressed in only in the veins (Wang et al., 1998).
As a result, our findings that the inhibition of Flk-1 or
Flt-1 modulates the proliferation of vascular endothelial
cells and the expression of ephrin-B2 in vascular endothelial cell suggests that the proportional VEGF signaling
mediated by means of Flk-1 and Flt-1 affects the arterial–
venous boundaries. Further investigation is called for to
clarify how VEGF and downstream signaling modulate
Fig. 5. A–H: Flk-1 expression is promoted by the inhibition of Flt-1.
B,C,G: There were more Flk-1–positive cells (C,G) in lung buds treated
with Flt-1 antisense oligodeoxynucleotide (AS-ODN) than those treated
with S-ODN (B). E,F,H: In contrast, there was no significant difference
in number of Flt-1–positive cells (F,H) in lung buds treated with Flk-1
AS-ODN compared with those treated with S-ODN (E). A–E: Immuno-
fluorescence images of lung buds [embryonic day (E) 11.5, cultured
for 48 hr] without ODN (A,D, ODN [2]), treated with S-ODN (B,E,
S-ODN), and treated with AS-ODN targeted to Flt-1 (C, AS-Flt-1) or
Flk-1 (F, AS-Flk-1) mRNA labeled with anti–Flk-1 (A–C) or anti–Flt-1
antibody (D–F). **P < 0.01. Scale bars 5 20 mm in A–F.
Fig. 6. A–N: Effects of antisense oligodeoxynucleotides (AS-ODNs)
directed to Flk-1 or Flt-1 mRNA on ephrin-B2 expression in culture.
A,B,E,F,I,J,M: There were few ephrin-B2–positive cells (B,F,J,M) in the
lung buds treated with Flk-1 AS-ODN in comparison to those treated
with S-ODN (A,E,I). C,D,G,H,K,L,N: In contrast, there were more abun-
dant ephrin-B2–positive cells (D,H,L,N) in Flt-1 AS-ODN–treated lung
buds than in S-ODN–treated buds (C,G,K). A–L: Immunofluorescence
images of ephrin-B2–positive cells (A–D, green), PECAM-1–positive
vascular endothelial cells (E–H, red), and merge images (I–L). *P <
0.05, **P < 0.01. Scale bars 5 20 mm.
VEGF RECEPTORS DURING LUNG DEVELOPMENT
971
Fig. 7. Schematic diagrams of the embryonic development of lung
vasculature (upper panels) and the inhibition of Flk-1 or Flt-1 (lower
panels). The embryonic development of the pulmonary vasculature is
divided into four stages. The inhibition of Flk-1 impaired branching of
capillaries, proliferation, and arterial differentiation in both vascular endothelial cells and their precursors. The inhibition of Flt-1 promotes
the branching, proliferation, and arterial differentiation of vascular endothelial cells by means of the up-regulation of Flk-1.
arterial–venous differentiation through ehrin-B2/EphB4
signaling at both the cellular and molecular levels.
organized capillary network. Despite the diffuse distribution of VEGF protein, the VEGF-receptors (Flk-1 and
Flt-1) were found to be spatiotemporally expressed and
they were thus found to play a role in both vascular endothelial cell proliferation and capillary branch morphogenesis. These results underscore the importance of Flk-1
and Flt-1 in the regulation of vascular endothelial cell development and pulmonary vascular branching. These
Conclusions and Future Perspectives
In this study, we focused on the role of Flk-1 and Flt-1
in vascular endothelial cell development and in the vascular remodeling involved in the transition from loose to
972
YAMAMOTO ET AL.
findings will hopefully provide new insight into the mechanism of normal pulmonary vascular development. Further investigations are necessary to elucidate the mechanisms behind the pathological conditions of such human
neonatal lung vasculature diseases as bronchopulmonary
dysplasia or alveolar capillary dysplasia.
ACKNOWLEDGMENTS
The authors gratefully acknowledge valuable assistance Mark A. Sussman, Heart Institute and Department
of Biology, San Diego State University, for his critical
review of this manuscript.
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