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Blood, 1 April 2002, Vol. 99, No. 7, pp. 2397-2407
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Vascular endothelial growth factor receptor Flt-1 negatively
regulates developmental blood vessel formation by modulating
endothelial cell division
Joseph B. Kearney,
Carrie
A. Ambler,
Kelli-Ann Monaco,
Natalie Johnson,
Rebecca G. Rapoport, and
Victoria L. Bautch
From the Program in Genetics and Molecular Biology,
Department of Biology, University of North Carolina at Chapel Hill,
Chapel Hill.
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Abstract |
Mice lacking the vascular endothelial growth factor (VEGF) receptor
flt-1 die of vascular overgrowth, and we are interested in how flt-1
normally prevents this outcome. Our results support a model whereby
aberrant endothelial cell division is the cellular mechanism resulting
in vascular overgrowth, and they suggest that VEGF-dependent
endothelial cell division is normally finely modulated by flt-1 to
produce blood vessels. Flt-1 / embryonic
stem cell cultures had a 2-fold increase in endothelial cells by day 8, and the endothelial cell mitotic index was significantly elevated
before day 8. Flt-1 mutant embryos also had an increased endothelial cell mitotic index, indicating that aberrant endothelial cell division occurs in vivo in the absence of flt-1. The
flt-1 mutant vasculature of the cultures was partially
rescued by mitomycin C treatment, consistent with a cell
division defect in the mutant background. Analysis of cultures at
earlier time points showed no significant differences until day 5, when
flt-1 mutant cultures had increased
 -galactosidase+ cells, indicating that the expansion of
flt-1 responsive cells occurs after day 4. Mitomycin C treatment
blocked this early expansion, suggesting that aberrant division of
angioblasts and/or endothelial cells is a hallmark of the
flt-1 mutant phenotype throughout vascular development.
Consistent with this model is the finding that expansion of platelet
and endothelial cell adhesion molecule+ and
VE-cadherin+ vascular cells in the
flt-1 mutant background first occurs between day 5 and day
6. Taken together, these data show that flt-1 normally modulates
vascular growth by controlling the rate of endothelial cell division
both in vitro and in vivo.
(Blood. 2002;99:2397-2407)
© 2002 by The American Society of Hematology.
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Introduction |
Blood vessels form by coordinating several
cellular processes, including cell division and morphogenesis (reviewed
in Folkman & D'Amore,1 Weinstein,2 and
Conway et al3). Some of the mitogenic signals that promote
division of endothelial cells and their precursors are known, but how
these signals are modulated to initiate cell divisions only when and
where they are needed is not known in detail. After blood vessels
initially form, maturation and remodeling steps involve the recruitment
of ancillary cells, such as smooth muscle and pericytes. These cells
and the extracellular matrix that is also produced can negatively
modulate endothelial cell division.4-8 However, modulators
of endothelial cell mitogenesis at the earliest stages of blood vessel
formation have not been identified.
The vascular endothelial growth factor (VEGF) signaling pathway is
clearly critical to both early endothelial cell division and
morphogenesis, and its regulation is complex (reviewed in Ferrara & Davis-Smyth9 and Neufeld et al10). Mouse
embryos lacking even one copy of the VEGF gene die in utero with severe vascular defects, and vascular development in differentiating embryonic
stem (ES) cells is compromised in VEGF-A+/ and
VEGF-A/ ES cells in a dose-dependent
manner.11-13 Moreover, even modestly elevated levels of
VEGF lead to vascular abnormalities,14 and large doses of
VEGF invariably severely compromise both vascular development and
neovascularization in adult organisms.15-17 These findings
suggest that VEGF signaling must be precisely controlled during
vascularization to result in proper vessels. The location and duration
of VEGF expression provide the first level of
control,18-21 but other components of the pathway are
likely to be involved in fine-tuning the signal.
Two high-affinity receptors, flk-1 and flt-1, participate in VEGF
signal transduction and are candidates to be involved in fine-tuning
mechanisms. Both receptors are membrane-spanning receptor tyrosine
kinases that bind VEGF with high affinity,22-26 but their effects on VEGF signaling are very different. Mice or ES cells lacking
flk-1 have little or no blood vessel formation, suggesting that many
downstream effects of VEGF on endothelial cells are mediated through
flk-1.27,28 Specifically, numerous studies show that VEGF
signaling through flk-1 produces a strong mitogenic signal for
endothelial cells.29-32
In contrast, VEGF binding to flt-1 does not produce a strong mitogenic
signal, and flt-1/ mice die at mid-gestation
with vascular overgrowth and disorganization.23,29,33 This
phenotype was reported to result from increased numbers of cells called
hemangioblasts that can give rise to both hematopoietic and endothelial
cells.34 However, invoking control of an early cell fate
switch as the exclusive cellular mechanism of flt-1 action is
inconsistent with evidence that flt-1 is expressed in mature
endothelial cells, including tumor vasculature.35-37 It is
also inconsistent with a molecular model of flt-1 action, suggesting that flt-1 can sequester VEGF ligand and, thus, modulate signaling through flk-1, because flk-1 signaling affects multiple endothelial processes, including cell division.38,39 Moreover, VEGF
addition to flt-1-expressing trophoblast cells inhibits cell division, and 2 recent studies using chimeric receptors suggested that flt-1 signaling may counteract the positive mitogenic signal from
flk.40-42
Thus, we asked if flt-1 could negatively modulate endothelial
mitogenesis developmentally, and to address this question we analyzed
the cellular mechanism responsible for the
flt-1/ phenotype in both ES cell cultures
and embryos. The flt-1 mutant ES cell cultures and embryos
had vascular overgrowth that was caused primarily by aberrant
endothelial cell division, and this deregulated mitogenesis in the
vascular lineage was seen throughout the stages of vascular
development. Thus, flt-1 acts early in vascular development to modulate
vessel formation by affecting the rate of cell division in embryonic
endothelial cells and their precursors.
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Materials and methods |
Cell culture and in vitro differentiation
Wild type (WT, +/+), hemizygous mutant
(flt-1+/), and homozygous mutant for the
targeted flt-1 mutation
(flt-1/)33 ES cells were
maintained and differentiated in vitro as attached cultures as
described previously.43
For mitomycin C treatment, ES cell cultures were differentiated to day
6, then incubated with mitomycin C (Sigma) at 30 µg/mL diluted in differentiation media for 2 hours at 37°C. After
incubation in fresh differentiation medium for 48 hours (to day 8),
cultures were fixed and stained with the appropriate antibodies. For
earlier times, cultures were incubated with mitomycin C as described
earlier on day 4 or day 5, then incubated in fresh medium for 24 hours (to day 5 or day 6) before fixation and staining.
Antibody staining and image analysis
ES cell cultures were rinsed in phosphate-buffered saline (PBS)
and fixed for 5 minutes in ice-cold methanol:acetone (50:50) or fresh
4% paraformaldehyde (for VE-cadherin staining). Fixed cultures were
reacted with antibodies as described previously.13,43 In
double-labeling experiments, cultures were first incubated with rabbit
anti--galactosidase or rabbit antiphosphohistone H3 antibodies and
the appropriate secondary, then blocked in staining media (3% fetal
bovine serum [FBS], 0.1% NaN3 in PBS) with 5% donkey
serum before the addition of rat antimouse platelet and endothelial
cell adhesion molecule (PECAM). In triple-labeling experiments, rabbit
polyclonal antiphosphohistone H3 incubation was followed by incubation
with rat antimouse PECAM and, subsequently, staining with the DNA dye
topro-3 (Molecular Probes) at 1:1000 for 5 minutes at room temperature.
All cultures were rinsed in PBS and viewed with an Olympus IX-50
inverted microscope by using epifluorescence or a Zeiss LSM 410 confocal microscope.
Primary antibodies and dilutions used were rat antimouse PECAM at
1:1000 (MEC 13.3; Pharmingen); rat antimouse intercellular adhesion
molecule 2 (ICAM-2) at 1:500 (3C4; Pharmingen), rabbit polyclonal
anti--galactosidase at 1:300 (Cappel Labs), rabbit polyclonal
antiphosphohistone H3 at 1:500 (Upstate Biotechnology), and rat
antimouse VE-cadherin at 1:100 (11D4.1; Pharmingen). Secondary antibodies and dilutions used were donkey antirabbit immunoglobulin G
(IgG; H + L) TRITC cross-absorbed at 1:100 (Jackson
Immunoresearch) for antiphosphohistone H3 and -galactosidase, donkey
antirat IgG (H + L) B-phycoerythrin cross-absorbed at 1:300
(Jackson Immunoresearch) for PECAM and ICAM-2, donkey antirat IgG
(H + L) fluorescein isothiocyanate (FITC) cross-absorbed at 1:100
(Jackson Immunoresearch) for PECAM, and goat antirat IgG (H + L)
Alexa 488 cross-absorbed at 1:100 (Molecular Probes) for PECAM and
VE-cadherin.
Quantitative image analysis of day 8 ES cell cultures reacted with the
appropriate antibodies was performed as previously described.13 Sequential nonoverlapping areas completely
covered with cells were photographed at ×10 magnification, so that the total area photographed per well was more than 60% of the well area.
For earlier time points, -galactosidase-stained wells were photographed, and only areas covered with cells were used for analysis.
Digital images were generated and analyzed by using Adobe Photoshop
(version 5.0, Adobe Systems). Quantitation of the stained area for each
image was performed by using an Image Processing Tool Kit (Rev. 2.1;
Reindeer Games, Asheville, NC). Stained area averages for each well
were calculated, and the average of 3 to 4 wells for each condition was
used to determine SD values.
-Galactosidase detection
-Galactosidase detection was performed by using a modified
protocol.44 Cultures were rinsed twice in 0.1 M phosphate
buffer (pH 7.3) and fixed with glutaraldehyde fix solution (0.2%
glutaraldehyde, 5 mM EGTA [pH 7.3], 2 mM MgCl2 in 0.1 M
phosphate buffer [pH 7.3]) for 5 minutes. After washing 3 times for 5 minutes with phosphate buffer, cultures were incubated for 3 hours (day
8 ES cultures) or 5 hours (early time course experiments) at 37°C in
X-gal staining solution (0.625 mg/mL X-gal; Sigma), 5 mM potassium
ferrocyanide, 5 mM potassium ferricyanide, in wash buffer (2 mM
MgCl2, 0.02% Nonidet-P40 in 0.1 M sodium phosphate buffer
[pH 7.3]), then rinsed and stored in wash buffer at 4°C.
RNA analysis
Total RNA was isolated from day 7 ES cell cultures by
centrifugation through a CsCl gradient.45 RNase protection
assays for PECAM were performed by using a modified
protocol.13,46 In vitro transcription of PECAM-dCPa (nt
1425-1904) was used to generate a 32P-labeled antisense
RNA probe. Overnight hybridization at 45°C with the PECAM probe and a
-actin internal control probe was followed by digestion with RNase A
and RNase T1. Protected fragments were then electrophoresed through a
5% acrylamide urea (8 mM gel) and quantified by using a PhosphorImager
(Molecular Dynamics).
Fluorescent-activated cell sorter analysis
Day 8 ES cell cultures were rinsed twice with PBS and
dissociated with 0.2% collagenase (Sigma; 0.15% type II, 0.05% type XI in PBS) for approximately 2 hours with repeated passage through a
20-gauge needle. The cells were rinsed in FBS/PBS (1:1), resuspended in
cold staining media (3% FBS + 0.01% sodium azide in PBS), and incubated on ice for 20 minutes. Cells were then incubated with 100 µg/mL biotin-coupled ICAM-2 antibody in staining medium for 45 minutes at 4°C. After 3 washes with cold staining medium, cells were
resuspended in staining medium with 25 µg/mL
streptavidin-phycoerythrin (Southern Biologicals) and incubated for 45 minutes at 4°C. After 3 washes with cold PBS, the cells were fixed
and stored at 4°C in 1% paraformaldehyde. Flow cytometry data were
collected with a Becton Dickinson FACSCAN.
Mitotic index calculations
WT and flt-1/ ES cell cultures were
differentiated in chamber slide wells (Nunc) to day 6 or 7, fixed, and
triple-labeled with rabbit antiphosphohistone H3, rat antimouse PECAM,
and the DNA binding dye topro-3. Slides were mounted in AquaPolymount
(LifeSciences). Confocal images were analyzed by using Adobe Photoshop
(version 5.0, Adobe Systems) software. Triple-labeled images were
counted in the following 4 ways: (1) the total number of cells per
field, (2) the total number of phosphohistone H3+ cells per
field, (3) the number of PECAM+ cells with endothelial
morphology per field, and (4) the number of
PECAM+/phosphohistone H3+ cells with
endothelial morphology per field. Endothelial mitotic indices were
calculated on a per field basis by dividing the number of
PECAM+, phosphohistone H3+ cells by the total
number of PECAM+ cells. Nonendothelial mitotic indices were
also calculated on a per field basis by dividing the number of
PECAM, phosphohistone H3+ cells by the total
number of PECAM cells. Data were collected from multiple
fields of multiple wells and averaged for each day.
Embryo immunohistochemistry
Flt-1+/ mice maintained on the CD-1
background were intercrossed to obtain embryos. Embryos were dissected
from the maternal decidua at day 8.5 (the morning of the plug is day
0.5), heads were removed and saved at  20°C for genotyping by using
a modification of a published protocol,33 and the rest of
the embryo was fixed in Serra fixative47 or cold 4%
paraformaldehyde at 4°C overnight. The embryos were dehydrated
through a methanol series and stored at 20°C in 100% methanol.
Embryos were embedded in paraffin, sectioned at 10 µm on a Zeiss
Microm, dewaxed in Histoclear, and rehydrated. Sections fixed in
paraformaldehyde were incubated in 0.02% Protease XXIV (Sigma) in PBS
for 4 minutes, then washed 3 times in PBS. After blocking in 0.25%
H2O2 in PBS for 15 minutes, primary antibody
(1:250 dilution in 5% goat serum/PBS) was added, and sections were
incubated overnight at 4°C in humidified chambers. After 3 washes in
PBS, secondary antibody (1:300 dilution of goat antirabbit or antirat
IgG-horseradish peroxidase [Accurate] in 5% goat serum/PBS) was
added, and incubation was overnight as before. After 3 washes in PBS,
sections were incubated in 3'-diaminobenzidine tetrahydrochloride
substrate to which 3 mg/mL NiSO4 was sometimes added (for
blue color) for 15 minutes. Slides were rinsed in PBS, incubated in a
1:10 000 dilution of DAPI (1 mg/mL stock) in H2O for 10 minutes, mounted using Glycergel (Dako), and visualized with a Nikon
Eclipse E800 microscope outfitted with DIC optics and epifluorescence.
To count mitotic endothelial nuclei, alternate sections were stained
with PECAM and phosphohistone H3. The DAPI-stained nuclei were used to
overlay digital images.
| |
Results |
Flt-1/ ES cell cultures have increased
vascularization
ES cells undergo a differentiation program in vitro that mimics
early murine yolk sac development, including primitive hematopoietic development and blood vessel formation.43,48-51
Hematoendothelial development begins when a mesodermally derived
hemangioblast population arises at days 2 to 3 of
differentiation,52 and angioblasts can also differentiate
directly from mesoderm. The endothelial cells of primitive blood
vessels are differentiated from angioblasts by coexpression of PECAM
and ICAM-2, both adhesion receptors of the immunoglobulin
superfamily.13,53,54 We initially
investigated the cellular mechanism of flt-1 in day 8 cultures, when
the PECAM+/ICAM-2+ vasculature is well established.
Flt-1+/ and flt-1/
ES cells were engineered so that Escherichia coli lacZ is
expressed under flt-1 regulatory control in the targeted
gene.33 These ES cells and WT (+/+) controls
were stained for -galactosidase activity or for PECAM expression at
day 8 (Figure 1). The
flt-1/ cultures had a dramatically expanded
-galactosidase expression domain compared with the
flt-1+/ cultures (Figure 1A). The
-galactosidase+ cells in the
flt-1/ cultures were found in large circular
sheets, with areas of normal-looking vasculature at the edge of the
sheets (Figure 1A,B, arrow). Immunofluorescent antibody staining for
PECAM, ICAM-2, or VE-cadherin showed a similar pattern in the
flt-1/ cultures (Figure 1B and data not
shown), suggesting that most of the -galactosidase-expressing cells
were endothelial cells. The -galactosidase- and antibody-stained
cells were elongated and interconnected, indicating that they were
endothelial cells. This criterion is important, because subsets of
hematopoietic cells can also react with the antibodies to PECAM or
ICAM-2. Only a ring of intensely -galactosidase+ cells
(Figure 1A, arrowheads) did not appear to stain for PECAM or ICAM-2 by
double-label immunofluorescent antibody staining (data not shown).
These cells were found in both WT and mutant cultures, and they reacted
with a flt-1 antisense RNA probe in the WT background (data not shown),
indicating that they are nonvascular flt-1-expressing cells.
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| Figure 1.
Flt-1/ ES cell cultures have increased
vascularization.
Day 8 differentiated flt-1/ (A-C),
flt-1+/ (D-F), and WT (G-I) cultures were
processed for -galactosidase detection (A,D,G) or reacted with an
antibody to PECAM (B,E,H). A and B show one quadrant of the relatively
large -galactosidase+ (A) or PECAM+ (B)
sheet of cells that characterizes the flt-1/
phenotype. In contrast, an extensive vascular plexus is found in both
flt-1+/ (E) and WT (H) ES cell cultures.
Arrowheads (A) outline an intensely stained
-galactosidase+ ring of cells that surrounds most of the
-galactosidase+ cells. Arrows (A,B) point to
flt-1/ vasculature that looks WT. (C,F,I)
Phase contrast images of PECAM-labeled fields in B,E,H. Magnification
is ×10.
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The increase in endothelial cells observed in mutant cultures was
quantitated in several ways (Figure 2).
RNase protection analysis of day 8 cultures with a PECAM antisense RNA
probe revealed that PECAM RNA levels were 2.5- to 3.3-fold higher in
flt-1/ cultures compared with WT cultures
(Figure 2A). Quantitative image analysis on day 8 ICAM-2-labeled
cultures used digital images of vascular immunofluorescence to
determine the percentage area stained, which approximates the amount of
vasculature (see "Materials and methods" section for detailed
protocols). Flt-1/ cultures exhibited nearly
a 2-fold increase in ICAM-2 staining area over WT, whereas
flt-1+/ cultures had essentially WT levels
(Figure 2B). ICAM-2 antibody-stained cultures were also processed for
fluorescent-activated cell sorting (FACS; Figure 2C).
Flt-1/-attached cultures contained a
population of ICAM-2+ cells that was significantly
increased over WT levels (compare 39% with 25%, respectively),
whereas flt-1+/ cultures had WT numbers of
ICAM-2+ cells. Similar FACS results were obtained with
antibodies to PECAM (data not shown). Taken together, these data show
that the lack of flt-1 results in increased numbers of vascular
endothelial cells.
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| Figure 2.
Flt-1/ ES cell
cultures have increased numbers of endothelial cells.
(A) RNase protection assay using an antisense PECAM RNA probe on
day 8 WT, flt-1+/, and
flt-1/ attached cultures. Protected
fragments were separated on a polyacrylamide-urea gel and quantified by
using a PhosphorImager. Protected PECAM signal was normalized to a
-actin signal, and the normalized PECAM band densities for
flt-1+/ and flt-1/
samples were compared with WT (+/+) samples. Sample 1 and
sample 2 are RNAs from separate differentiations. Each bar is the
average of 3 experiments performed on a particular sample. (B)
Quantitative image analysis of the ICAM-2+ area on day 8 WT
(+/+), flt-1+/, and
flt-1/ attached cultures. Each bar
represents the average area stained with ICAM-2 antibody from 3 wells.
This experiment was repeated (data not shown), and similar quantitative
trends were obtained. (C) Fluorescent cell sorting of ICAM-2-labeled
day 8 WT (+/+), flt-1+/, and
flt-1/ ES cell cultures. The plots in dotted
lines are controls without primary antibody.
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Lack of flt-1 leads to increased endothelial cell division
To investigate the cellular mechanism(s) responsible for the
increased vascularization seen in the absence of flt-1, the hypothesis that flt-1/ endothelial cells have a higher
rate of cell division than WT endothelial cells was tested. Day 6 and
day 7 ES cell cultures were labeled with antibodies to the vascular
marker PECAM and to the mitotic marker phosphohistone
H3,55 then stained with a DNA-binding dye (topro-3; Figure
3). Visual observation suggested that day
6 and day 7 flt-1/ ES cell cultures had more
PECAM+ cells that colabeled with the antiphosphohistone H3
antibody than WT controls (compare Figure 3A,C with B,D and E with
F).
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| Figure 3.
Flt-1/ ES cell cultures have mitotic
endothelial cells.
Day 7 (A-D) or day 6 (E,F) WT (A,C,E), and
flt-1/ (B,D,F) attached cultures were
labeled with antibodies to PECAM (green) and phosphohistone H3 (red),
then stained with the nuclear marker topro-3 (blue). The arrowhead (B)
shows a phosphohistone H3+ nonendothelial cell
(PECAM), whereas the arrow (B) points to a phosphohistone
H3+ endothelial cell (PECAM+). Notice the
increase in phosphohistone H3+/PECAM+ cells in
flt-1/ cultures relative to WT cultures. All
panels are confocal images at ×40
magnification.
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To quantitate the apparent increase in mitotic PECAM+ cells
in flt-1/ cultures, confocal images from day
6 or day 7 fixed cultures processed as in Figure 3 were used to
calculate cell counts and mitotic indices for both endothelial and
nonendothelial cell populations (Table 1
and Figure 4; see "Materials and
methods" section for details). In all cases endothelial cells of the
flt-1/ cultures had a higher mitotic index
than WT endothelial cells. To control for differential growth rates, a
nonendothelial cell mitotic index was obtained for each experiment
(Table 1). There was little difference between WT and
flt-1/ nonendothelial cell mitotic indices
within a given experiment, in contrast to increases in the
flt-1/ endothelial cell mitotic index. Each
endothelial cell mitotic index was normalized to its companion
nonendothelial cell mitotic index (Figure 4; Table 1, far right
column). Day 6 flt-1/ cultures had
normalized endothelial cell mitotic indices that were 3- to 4-fold
higher than normal, and similar but less dramatic trends were observed
in day 7 cultures (Figure 4, compare black bars with gray bars). These
results indicate that the increased vascularization seen in day 8 flt-1/ ES cell cultures is caused, at least
in part, by an increased endothelial cell division rate in the absence
of flt-1.
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Table 1.
Comparison of endothelial and nonendothelial mitotic
indices in wild type and flt-1/ embryonic
stem cell cultures
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| Figure 4.
Flt-1/ ES cell cultures have
an elevated endothelial cell mitotic index.
Days 6 and 7 WT (+/+) and flt-1/
triple-labeled images were used to calculate nonendothelial cell and
endothelial cell mitotic indices for 2 separate differentiation
experiments (Table 1). Endothelial cell mitotic indices were expressed
as a percentage of the nonendothelial cell mitotic index calculated for
each experimental condition. The dotted black line represents the
nonendothelial mitotic index for each experiment converted to 100%,
and it was used as a baseline for comparison of endothelial
mitotic indices.
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If aberrant endothelial cell division contributes to the
flt-1 mutant phenotype, then blocking cell division during
ES cell differentiation may affect the phenotype. Thus, day 6 ES cell cultures were treated with the replication inhibitor mitomycin C before
incubation for an additional 2 days (Figure
5). Untreated flt-1/ cultures fixed on day 6 had slightly
increased numbers of PECAM+ cells compared with day 6 WT
cultures (compare Figure 5A with B). Treated day 8 flt-1/ had half as much vasculature as
untreated genotype-matched controls, accompanied by a dramatic decrease
in the labeling of nuclei with antiphosphohistone H3 (compare Figure 5D
with F,H). In some cases, mitomycin C-treated
flt-1/ vasculature at day 8 was branched and
appeared WT in morphology (Figure 5G), suggesting that blocking cell
division during days 6 to 8 of differentiation can partially compensate
for the lack of flt-1 in vascular development. Mitomycin C treatment
also affected vascular growth in WT cultures, which is predicted
because blood vessel formation requires endothelial cell division. The
treated WT cultures had 2- to 3-fold less vasculature and less
branching than untreated controls (compare Figure 5C with E,H). Thus,
treatment with mitomycin C, an inhibitor of replication, partially
rescues the flt-1 mutant vascular phenotype.
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| Figure 5.
Mitomycin C treatment partially rescues the
flt-1/ vascular phenotype.
Day 6 ES cell cultures were fixed (A,B), left untreated (C,D), or
treated with mitomycin C (E-G). Some cultures (C-G) were differentiated
for an additional 48 hours. Cultures were labeled with an antibody to
PECAM (green), and some cultures (C-F) were also labeled with the
mitotic marker antiphosphohistone H3 (red). Notice the abundance of
phosphohistone H3-labeled figures in untreated (C-D) cultures
compared with treated (E-F) cultures. (G) Example of a treated
flt-1/ culture that morphologically
resembled WT vasculature. (H) Quantitative image analysis of the
PECAM+ area of day 8 WT (+/+) and
flt-1/ (/)
cultures treated with mitomycin C (red) or left untreated (green). Each
bar represents the average stained area from at least 3 wells stained
with PECAM antibody. Magnification was ×10 except C (×20) and G
(×4).
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Flt-1 mutation affects division of vascular precursor
cells
To determine when the flt-1 mutation first affects vascular
development, we investigated earlier time points of ES cell
differentiation. To establish when cells expressing lacZ under
control of the flt-1 promoter were first affected by the lack of flt-1
protein, we analyzed an early time course of ES cell differentiation.
We plated cells directly after dispase treatment, then processed wells
of each genotype for lacZ expression on days 2 to 6 of differentiation (Figure 6). The percentage of
lacZ-expressing cells was equivalent between
flt-1+/ and flt-1/
cultures on days 2 to 4, and only on day 5 was there a significant increase in the percentage of lacZ-expressing cells in the flt-1 mutant
background (Figure 6A). To determine if this expansion was the result
of aberrant cell division, wells were treated with mitomycin C on day 4 or day 5, then compared with control untreated wells 24 hours later.
Day 5 flt-1/ mutant cultures treated with
mitomycin C 24 hours earlier had fewer lacZ-expressing cells than
paired untreated controls (compare Figure 6C with D). The day 5 mitomycin C-treated wells were, in fact, similar to untreated wells
fixed at day 4 (compare Figure 6B with C). Day 6 flt-1/ mutant cultures treated with
mitomycin C 24 hours earlier also had fewer lacZ-expressing cells than
paired untreated controls (compare Figure 6E with F). These results
show that the earliest expansion of lacZ-expressing cells in the
flt-1/ mutant cultures can be inhibited by
mitomycin C, suggesting that the expansion results from aberrant
cell division.
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| Figure 6.
Mitomycin C-sensitive expansion of -galactosidase-expressing cells
in flt-1/ ES cell cultures at earlier
times.
(A) Quantitative image analysis of the -galactosidase+
areas of flt-1+/ (light blue bars) and
flt-1/ (dark blue bars) ES cell cultures on
days 2 to 5 of in vitro differentiation. For days 2 and 3, the bars
represent the average -galactosidase+ area for 9 individual attached ES cell clumps. For days 4 and 5, the bars
represent the average -galactosidase+ area for 2 culture
wells. The asterisk (*) indicates significance at
P < .001. (B-F) Days 4 to 6 flt-1/ ES cell cultures untreated (B,D,F) or
treated with mitomycin C (C,E) and stained for -galactosidase
activity. (B) Day 4 flt-1+/ culture. (C) Day 5 flt-1/ culture treated on day 4 with
mitomycin C. Note decrease in stained area relative to (D) untreated
day 5 flt-1/ culture. (E) Day 6 flt-1/ culture treated on day 5 with
mitomycin C. Note decrease in stained area relative to (F) untreated
day 6 flt-1/ culture. Original
magnification, ×20.
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Because both endothelial cells and a nonendothelial cell population
express flt-1 promoter-driven -galactosidase, we investigated the
expression of several vascular markers in the ES cell cultures. Cultures were stained with PECAM or VE-cadherin from days 2 to 6 of
differentiation (Figure 7), because both
markers are expressed early in vascular development. PECAM was
expressed throughout the time course, but before day 5 only clumps of
PECAM+ cells were seen, and no significant differences were
seen among the different genotypes (data not shown). By day 5 both WT
and flt-1+/ cultures had some areas of
PECAM+ vasculature, but surprisingly the
flt-1/ mutant day 5 cultures had few
PECAM+ cells and most were still in clumps (Figure 7A-C).
By day 6 all cultures had PECAM+ vasculature, and the
flt-1/ mutant cultures had as much or more
PECAM+ vessels compared with WT or
flt-1+/ cultures (Figure 7G-I). Treatment of
flt-1/ cultures from days 5 to 6 with
mitomycin C reduced the number of PECAM+ cells (data not
shown). VE-cadherin+ cells were not seen in any cultures
until day 5 (data not shown). Similar to the PECAM pattern, on day 5 WT
and flt-1+/ cultures had
VE-cadherin+ vasculature, whereas the
flt-1/ mutant cultures had only a few
VE-cadherin+ cells that were not organized into vessels
(Figure 7D-F). By day 6 cultures of all genotypes had
VE-cadherin+ vessels (Figure 7J-L). These results show that
flt-1/ mutant cultures did not have
expansion of either PECAM+ or VE-cadherin+
vascular cells until between days 5 and 6 of differentiation, when
expansion of both -galactosidase-expressing cells and
PECAM-expressing cells was sensitive to mitomycin C.
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| Figure 7.
Expression of vascular markers in differentiating ES cell cultures.
Wt (+/+) (A,D,G,J), flt-1+/
(B,E,H,K), and flt-1/ (C,F,I,L) ES cell
cultures were fixed on day 5 (A-F) or day 6 (G-L) and labeled with
antibodies to PECAM (A-C,G-I) or VE-cadherin (D-F,J-L), and the
appropriate fluorescent-labeled secondary antibody. Arrows (C,F) point
to sparse PECAM+ and VE-cadherin+ cells in day
5 flt-1/ cultures. Original magnifications
×20, except D-F at ×40.
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Flt-1/ embryos have increased
mitoses
To determine if the aberrant endothelial cell division seen in the
absence of flt-1 during ES cell differentiation also occurred in vivo,
day 8.5 embryos were stained with the antiphosphohistone H3 antibody
(Figure 8). The
flt-1/ mutant embryos had numerous mitotic
nuclei in several vascular areas, including the lining of yolk sac
blood islands (Figure 8B,C,E,F) and the allantois (Figure 8F). In
contrast, nonmutant embryos had far fewer mitotic nuclei in those areas
(Figure 8A,D). The increase in mitotic nuclei was specific to vascular
areas in vivo, because embryonic structures such as the neural tube and
somites had roughly equivalent numbers of mitotic nuclei regardless of
the genetic background (data not shown). Digital overlays of alternate
sections stained with PECAM and phosphohistone H3 (Figure 8D-F) were
used to calculate the endothelial mitotic indices in vivo. The
endothelial mitotic index of flt-1/ embryos was 2.8%
(n = 1270), double that of WT+/+ embryos whose
endothelial mitotic index was 1.4% (n = 425). Thus, the aberrant
endothelial cell division documented during ES cell differentiation in
the absence of flt-1 is also a hallmark of the mutant phenotype in
vivo.
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| Figure 8.
Flt-1 / embryos have increased mitoses in
endothelial cells.
Transverse sections of day 8.5 embryos were processed for
immunohistochemistry by using antiphosphohistone H3 to detect mitotic
nuclei (A-C), and overlays of adjacent sections were processed
individually (see "Materials and methods" section) for
immunohistochemistry with antiphospohistone H3 (red), anti-PECAM
(green), and DAPI (blue) (D-F). Visualization of yolk sacs of
flt-1+/ (A) or WT (+/+) (D)
embryos that were phenotypically normal showed few mitotic nuclei in
vascular areas (arrow in D). In contrast,
flt-1/ embryos (B,C,E,F) exhibited vascular
overgrowth and numerous mitotic nuclei (red; E,F) in PECAM+
regions (green; E,F) of the yolk sac and allantois (F, left part of
panel). (A-C) Asterisks denote the lumina of blood islands in the yolk
sac, and arrows point to mitotic nuclei abutting the endoderm with the
long axis perpendicular to the long axis of the endoderm cells, a
characteristic of dividing endothelial cells. In contrast, the
arrowhead in C points to a mitotic nucleus in the endoderm with the
long axis parallel to the long axis of the endoderm cells, a
characteristic of dividing endoderm. The arrowhead in B points to a
mitotic nucleus of unknown cell type. (D-F) Arrows point to mitotic
nuclei of PECAM+ cells. En, visceral endoderm of
the yolk sac.
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Discussion |
Our data support a model whereby flt-1 normally affects early
vascular development by negatively modulating cell division in the
vascular lineage. The identification of this cellular mechanism of
flt-1 action suggests that flt-1 is critical for the fine tuning of
VEGF-mediated vessel growth that is required to form proper blood
vessels. It also strongly suggests that flt-1 may affect blood vessel
formation in similar ways in both the embryo and the adult. Embryos and
differentiated ES cells lacking flt-1 have increased vascularization
and numbers of endothelial cells accompanied by an increased
endothelial cell mitotic index. In contrast, the nonendothelial cell
mitotic index is similar in both genetic backgrounds, indicating that
the increased mitotic rate in the flt-1/
background is endothelial cell specific.
The ability of mitomycin C to partially rescue the
flt-1/ vascular phenotype further supports
the conclusion that deregulated endothelial cell division is
responsible for the flt-1 mutant phenotype. The WT cultures
were also affected, which was expected because endothelial cell
division is a critical component of normal blood vessel
formation.56 A caveat is that mitomycin C inhibits division in all cells, so lack of division in nonendothelial cells could indirectly affect the endothelial cell phenotype. This scenario cannot be ruled out, but the increased endothelial mitotic index in the
flt-1 mutant background and its diminution with mitomycin C
suggest that a substantial part of the rescue is likely to result from
direct effects on endothelial cell division. This model can be more
precisely tested by expressing genes that modulate cell division under
the control of endothelial-specific regulatory sequences in the mutant
ES cells.
Flt-1 modulates cell division in the vascular lineage at the earliest
stages of vascular development. The first documented difference in ES
cell cultures was at day 5, when flt-1/
mutant cultures had more cells expressing -galactosidase under control of the flt-1 promoter than flt-1+/
cultures. The exact identity of these cells is unclear because we have
identified a nonvascular, flt-1-expressing cell population in ES cell
cultures, and several cell types such as trophoblasts and
monocyte/macrophages express flt-1 in vivo.40,57-59
However, because endothelial cells also express flt-1, it is likely
that at least a subpopulation of these cells are vascular precursor cells. In any case, the expansion of -galactosidase-expressing cells in the flt-1/ mutant background could
be blocked by mitomycin C from days 4 to 5 onward, indicating that the
expansion of this cell population resulted from aberrant cell division.
Interestingly, vascular cells expressing PECAM and/or VE-cadherin were
much less prevalent in the flt-1/ mutant
cultures on day 5, suggesting that different subpopulations of vascular
precursor cells may be affected by the flt-1 mutation at different
times. The expansion of PECAM+ and/or
VE-cadherin+ vascular cells was not evident until day 6 in
the flt-1/ mutant background, and this
expansion was also blocked by mitomycin C. Thus, flt-1 has a major role
in modulating cell division in the vascular lineage starting at days 4 to 5 of ES cell differentiation, just before formation of the first
primitive blood vessels.
Other processes can also affect the number of endothelial cells,
including cell fate decisions and programmed cell death. Appreciable
endothelial cell death is not observed during days 5 to 8 of normal ES
cell differentiation (V.L.B., unpublished observation), so
inhibition of apoptosis is unlikely to make a major contribution to the
flt-1 mutant phenotype. Our results do not formally exclude that, in
addition to an effect on vascular cell division, flt-1 may alter cell
fate by affecting hemangioblast formation,34 but our
results are not consistent with this model. We see no significant
differences between normal and mutant cultures until day 5, well beyond
the peak of hemangioblast formation at days 2.5 to 3.0.52
In the hemangioblast study, increased PECAM and -galactosidase
staining during differentiation of flt-1/
EBs was interpreted as increased hemangioblast numbers, but the lack of a definitive hemangioblast marker makes it impossible to
distinguish between hemangioblasts, angioblasts, and differentiated endothelial cells using these criteria. Moreover, in our hands the
expansion of the vascular lineage was blocked by mitomycin C at its
earliest detection on days 4 to 5, suggesting that the major effect of
the flt-1 mutation on vascular growth results from aberrant cell division.
The identification of flt-1 as an early modulator of cell division in
vascular development is consistent with several elegant studies showing
that flt-1 affects endothelial cell mitogenesis in cultured endothelial
cells.31,41,42 Extending this model of flt-1 action to the
earliest stages of development has several implications. First, it
suggests that deregulation of proliferation can be sufficient to
disrupt developmental processes. Other recent investigations of the
role of the cell cycle in development support this
hypothesis.60 Second, the data suggest that flt-1 can
modulate the endothelial cell cycle developmentally by affecting one or more molecular signaling pathways, although which pathways are affected
is not entirely clear. Deletion of the flt-1 tyrosine kinase domain
does not disrupt vascular development,58 suggesting that
signaling through this domain is not necessary for flt-1 to affect the
endothelial cell cycle developmentally. Signaling through flk-1 does
produce a strong endothelial mitogenic signal, and flk-1 selective
inhibitors partially rescue the flt-1/
phenotype in ES cell cultures (D. Roberts and V.L.B., unpublished results). This finding suggests that flt-1 affects vascular development at least in part by modulating VEGF-mediated flk-1 signaling, and this
modulation could occur in several ways.
A soluble form of flt-1, sflt-1, is expressed during
development61 and ES cell differentiation (J.B.K. and
V.L.B., unpublished results), and it can inhibit VEGF-dependent
endothelial cell division.38,39 Thus, sflt-1 can bind VEGF
and prevent ligand-induced dimerization of the flk-1 receptor. The
full-length receptor can also theoretically form an inactive
heterodimer with flk-1, as suggested by a recent study using chimeric
receptors.41 In addition, ligand engagement of flt-1 may
modulate flk-1 signaling at points downstream in the signal
transduction pathway. This model is supported by the inhibitor
sensitivity of chimeric receptors and a study implicating nitric oxide
as a mediator of flt-1 effects on the flk-1 mitogenic pathway.42,62 Importantly, these models of flt-1 action
are not mutually exclusive, and it is likely that flt-1 uses some combination of these actions to modulate endothelial cell division developmentally. The identification of the cellular mechanism of flt-1
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Abstract
Introduction