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Blood, Vol. 95 No. 6 (March 15), 2000:
pp. 1979-1987
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Characterization of the vasculogenic block in the absence
of vascular endothelial growth factor-A
Victoria L. Bautch,
Sambra D. Redick,
Aaron Scalia,
Marco Harmaty,
Peter Carmeliet, and
Rebecca Rapoport
From the Department of Biology and the Program in Molecular Biology
and Genetics, University of North Carolina at Chapel Hill, Chapel Hill,
NC, and the Center for Transgene Technology and Gene
Therapy, KU Leuven, Leuven, Belgium.
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Abstract |
Vascular endothelial growth factor (VEGF) signaling is required for
both differentiation and proliferation of vascular endothelium. Analysis of differentiated embryonic stem cells with one or both VEGF-A
alleles deleted showed that both the differentiation and the expansion
of endothelial cells are blocked during vasculogenesis. Blood island
formation was reduced by half in hemizygous mutant VEGF cultures and by
10-fold in homozygous mutant VEGF cultures. Homozygous mutant cultures
could be partially rescued by the addition of exogenous VEGF. RNA
levels for the endothelial adhesion receptors ICAM-2 and PECAM were
reduced in homozygous mutant cultures, but ICAM-2 RNA levels decreased
substantially, whereas PECAM RNA levels remained at hemizygous levels.
The quantitative data correlated with the antibody staining patterns
because cells that were not organized into vessels expressed
PECAM but not ICAM-2. These PECAM+ cell clumps accumulated
in mutant cultures as vessel density decreased, suggesting that they
were endothelial cell precursors blocked from maturation. A subset of
PECAM+ cells in clumps expressed stage-specific embryonic antigen-1
(SSEA-1), and all were ICAM-2( ) and CD34(), whereas vascular
endothelial cells incorporated into vessels were PECAM(+),
ICAM-2(+), CD34(+), and SSEA-1(). Analysis of flk-1 expression
indicated that a subset of vascular precursor cells coexpressed PECAM
and flk-1. These data suggest that VEGF signaling acts in a
dose-dependent manner to affect both a specific differentiation step
and the subsequent expansion of endothelial cells.
(Blood. 2000;95:1979-1987)
© 2000 by The American Society of Hematology.
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Introduction |
Our understanding of mammalian vascular development is
far from complete. Mesoderm-derived cells differentiate into
endothelial cells that coalesce into blood vessels in a process called
vasculogenesis, and the sprouting of endothelial cells from vessels
produces new vessels in a process called angiogenesis.1-3 A
relationship between the vascular and hematopoietic lineages through a
common precursor cell, the hemangioblast, has long been
hypothesized.1,4 Immature vascular precursor cells are
called angioblasts, but in most cases the angioblast is operationally
defined as a cell that participates in vasculogenesis. Nevertheless,
several markers are consistently associated with embryonic stem (ES)
cell-derived precursor cells having vascular potential, including the
vascular endothelial growth factor (VEGF) receptor flk-1, the cell
adhesion receptor platelet endothelial cell adhesion molecule (PECAM),
and the adhesion molecule VE-cadherin.5-7 These molecules
also have in vivo expression patterns that are consistent with their
being early markers of vascular lineage.8-12 However, the
paucity of early precursor cells available from mouse embryos makes
functional analysis of these putative precursor cells difficult.
VEGF signaling is critical for blood vessel formation during
development (reviews13-15). VEGF is a 45-kd homodimer
produced at sites of vasculogenesis and angiogenesis, and alternative
splicing results in 3 different isoforms in the mouse. The homodimer of VEGF-164 is the most active form, and its properties include
mitogenesis, chemotaxis, and permeability for endothelial cells. VEGF
binds to 2 high-affinity receptors, flk-1 (VEGFR-2) and flt-1
(VEGFR-1), that are expressed in endothelium. Recently, a third
molecule that binds VEGF with high affinity was identified as
neuropilin-1, a receptor that also signals in the nervous system
through a different ligand.16 Available data suggest that
neuropilin-1 acts as a coreceptor with flk-1 in vascular tissues.
Expression patterns of receptors and ligands suggest that VEGF may be a
highly specific mediator of blood vessel formation in
vivo,8,17,18 and analysis of targeted mutations in the mouse supports this hypothesis. Both flk-1 and flt-1 receptor mutations
are recessive embryonic lethals at days 8.5 to 9.5 of gestation.19,20 The flk-1 mutation severely impairs
vasculogenesis and hematopoiesis, whereas the flt-1 mutation affects
vascular organization. A targeted mutation in VEGF confirmed that this ligand is necessary for vascular development and showed a surprising dosage effect because embryos that were hemizygous for the
VEGF-targeted mutation died in utero with vascular
defects.21,22 The importance of flk-1 in developmental
blood vessel formation and hematopoiesis was further shown in a chimera
analysis with flk-1/ES cells.23 Although
these data suggest that VEGF signaling through the flk-1 receptor is
responsible for vasculogenesis and hematopoiesis, the recent
identification of VEGF-related molecules that can bind flk-1,
flt-1,24-27 , or both, makes it important to analyze the effects of the lack of VEGF on developmental processes.
ES cell differentiation in vitro has provided a tool to study specific
aspects of mammalian development (review28). ES cell differentiation in serum without added factors recapitulates many aspects of yolk sac development, including vascular and hematopoietic differentiation in the context of other yolk sac cell
types.5,29-32,33 This system was exploited to
identify early hematopoietic precursor cells and putative
hemangioblasts with vascular and hematopoietic potential.34,35 Differentiation of ES cells with specific
mutations indicates that defects in hematopoiesis and vascular
development are recapitulated in vitro. Differentiation of
flk-1/ES cells revealed the expected defects in
vasculogenesis, but primitive erythropoiesis was not compromised as it
is in vivo.23,36 The authors suggested that VEGF signaling
may be necessary in vivo for the migration of precursors to the yolk
sac environment, whereas in vitro the signals are produced in the
vicinity of the precursor cells.
To further elucidate the role of VEGF signaling in vascular development
in a system with the potential for functional analysis, we analyzed
VEGF mutant ES cells differentiated in vitro. Vascular development was
compromised in a dose-dependent manner and could be partially rescued
by the exogenous addition of VEGF. A population of cells that expressed
flk-1 but formed blood islands inefficiently accumulated in the VEGF
mutants, and this population partially overlapped with a second
population of cells that accumulated and expressed the cell adhesion
receptor PECAM. The PECAM+ and flk-1+ cells were negative for several
other vascular markers, including intercellular adhesion molecule-2
(ICAM-2) and CD34. These findings suggest that VEGF signaling is
required for the transition of flk-1+ and PECAM+ cells to vascular
endothelial cells that express ICAM-2 and CD34.
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Materials and methods |
Cell culture and in vitro differentiation
ES cells wild-type (wt, +/+), hemizygous mutants for the targeted
VEGF mutation (het, +/), and homozygous mutants for the targeted
VEGF mutation (null, /)21 were cultured with
5637 cell-conditioned medium as a source of leukemia inhibitory factor. They were differentiated by a modification of a protocol for embryoid body differentiation that allows for reattachment of the embryoid bodies to tissue culture plastic during the
differentiation.32 In vitro differentiation in attached
cultures was commenced by treatment with Dispase, 3 days in suspension
culture in differentiation media (DMEM-H, 20% fetal bovine serum (FBS;
lot tested), 150 µm  -thioglycerol, and 50 µg/mL gentamicin), and
transfer to 24-well tissue culture plates. Differentiation cultures
were fed every 48 hours, and in some cases the medium was supplemented
with human rVEGF-165 (R&D Systems, Minneapolis, MN) at 15 ng/mL at each
feeding from day 3 to day 8.
Antibody staining
Antibody staining was performed using a modified
protocol.32 Cultures were rinsed in PBS and fixed in the
appropriate fixative for 6 minutes ice-cold methanol:acetone (50:50)
for PECAM, ICAM-2, SSEA-1, and flk-1 antibodies, and fresh 4%
paraformaldehyde for CD34 antibody. PECAM/CD34 double stains were fixed
in paraformaldehyde, and all others were fixed in methanol:acetone.
Fixed cultures were blocked in staining media (3% FBS, 0.1%
NaN3 in PBS), incubated in primary antibody for 1 to 2 hours at 37°C, and rinsed in staining media. Secondary antibody was
incubated for 1 hour at 37°C. For double-labeling experiments, the
first reaction was followed by incubation with a rabbit anti-PECAM
antibody (gift of Beat Imhof) followed by the appropriate secondary
antibody. PECAM and flk-1 were double labeled by staining first
with rat antimouse PECAM, followed by staining with rabbit antimouse
flk-1. Cultures were rinsed in PBS and viewed with an
Olympus IX-50 inverted microscope using epifluorescence.
Primary antibodies and dilutions used were: rat antimouse PECAM at
1:1000 (Mec 13.3; Pharmingen, San Diego, CA); rat antimouse ICAM-2 at
1:500 (3C4; Pharmingen); rat antimouse CD34 at 1:500 (RAM34;
Pharmingen); mouse antimouse SSEA-1 at 1:50 (MC-480; Developmental Studies Hybridoma Bank); purified rabbit polyclonal
antimouse PECAM at 1:500; and rabbit polyclonal antimouse flk-1 at 1:50 (sc-504; Santa Cruz Biotechnology, Santa Cruz, CA). Secondary antibodies used were donkey antirat B-phycoerythrin (BPE)
cross-absorbed at 1:300 (712-106-150; Jackson Immunoresearch, West
Grove, PA); goat antimouse IgM Fab fragment-BPE cross-absorbed at 1:500
(115-106-075; Jackson Immunoresearch) for MC-480; goat antirabbit IgG
(H + L)-fluorescein isothiocyanate (FITC) cross-absorbed at 1:500
(4050-02; Southern Biotechnology Associates, Birmingham, AL) for
polyclonal PECAM; and donkey antirabbit IgG (H + L) TRITC
cross-absorbed at 1:100 (711-025-152; Jackson Immunoresearch) for
polyclonal flk-1. Py-4-1 endothelial cells37 and NIH 3T3
fibroblast cell lines were used as positive and negative controls,
respectively, for PECAM, ICAM-2, CD34, and flk-1. For SSEA-1
reactivity, ES cells were the positive control and NIH 3T3 cells were
the negative control. In all cases controls with no primary antibody or
an irrelevant isotype-matched control did not produce signal over
background (data not shown).
Imaging and quantitation
Embryonic stem cells were plated onto a 48-well tissue culture dish,
maintained as described above, and fixed on day 8 of differentiation.
After staining with the appropriate primary and secondary antibodies,
good-quality wells were photographed using Tmax 400 film at
a fixed exposure time at 10× magnification. Sequential frames
were set up to provide 7 nonoverlapping frames with 100% cell coverage
from each well. This strategy allowed for the analysis of more than
60% of each well area. Each frame was scanned using a SprintScan 35 (Polaroid, Cambridge, MA), and the resultant digital image was manually
modified in Adobe Photoshop 3.0 to remove the background. The modified
image was analyzed using an Image Processing Tool Kit
(Rev. 2.1; Reindeer Games, Asheville, NC) that allowed quantitation of the stained area. The average for each well was calculated, the numbers for 2 to 4 wells of each condition were averaged, and the SD was calculated. The relative abundance of PECAM+
aggregates was determined by visual examination of multiple PECAM-stained wells of each genotype in several independent differentiations.
RNA analysis
Total RNA was isolated by centrifugation through a CsCl
cushion38 and analyzed using a modified RNase protection
assay.39 32P-labeled antisense RNA probes were
generated by in vitro transcription of the following cloned gene
fragments: p4-21L1 (flk-1, nt 2399-2687) (gift of D. Dumont); clone 13 (flt-1, nt 2321-2598), Te2 (tie-2, nt 2057-2435), VEGFCon (VEGF, nt
1-400, a subclone of VEGFAllie) (all gifts of K. Peters);
I-2700 (ICAM-2, nt 556-817), and PECAM-dCPa (PECAM, nt
1425-1904). After overnight hybridization at 45°C with a specific
probe and  -actin as an internal control, the reactions were digested
with RNase A and RNase T1. The protected fragments were electrophoresed
through a 5% acrylamide/8 mol/L urea gel, then visualized and
quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
In situ hybridization
In situ hybridization was performed by modification of a standard
protocol used for whole-mount hybridization.40 RNA probes were synthesized using p421L1 as a template for flk-1 and PECAM-dCP as
a template for PECAM. PECAM-dCP was a reverse transcription-polymerase chain reaction amplification product of 660 bp from nt 1244 to nt 1904 generated from Py-4-1 endothelial cell RNA using a TA cloning kit
(Invitrogen, Carlsbad, CA) (M. Inamdar and V. L. Bautch, unpublished results). Cultures from days 8 or 9 were fixed in fresh 4% paraformaldehyde for 1 hour and dehydrated through a methanol:PBS series for storage at 20°C. Wells were
rehydrated, incubated first in 6% H2O2 in PBT
(PBS + 0.1% Tween-20) for 30 minutes, incubated in proteinase K (3 µg/mL in PBT) for 10 minutes, and then incubated in fresh glycine (2 mg/mL in PBT) for 4 minutes. All incubations were followed by three
5-minute washes in PBT. Wells were refixed in 0.2% glutaraldehyde and
4% paraformaldehyde in PBT for 8 minutes, washed, and prehybridized
for 2 to 3 hours at 70°C as described. Wells were hybridized in
hybridization buffer with denatured probe (2 µL of a 50-µL probe
reaction/0.25 mL hybridization buffer/well). An initial 3-hour
incubation at 80°C was followed by a 36-hour incubation at
69°C. Posthybridization washes were as described at 65°C. RNase
treatment, blocking incubation, antibody reaction, washes, and
substrate development were all as described. Cultures were developed
for 25 to 36 hours, then postfixed and stored in PBT at
4°C.
Statistical analysis
The average area stained (imaging) or protected counts (RNA) were
calculated from multiple wells, multiple experiments, or both. Averages
for each genotype were compared to wild-type values using the Student
t test. For the VEGF rescue experiments, the rescue average was
compared to the average of unrescued wells of the same genotype using
the Student t test. Significant differences are denoted by
asterisks in the appropriate figures.
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Results |
Vascular development is compromised in the absence of VEGF
In vitro differentiation of wild-type (+/+, wt), hemizygous
(+/, het), and homozygous (/, null) VEGF mutant ES
cells21 to day 8 revealed defective vascular development in
both hemizygous and null VEGF mutant cultures (Figures
1 and
2). Cultures were analyzed
by immunofluorescent antibody staining for expression of the vascular
adhesion molecule ICAM-2 (Figures 1A-1C). ICAM-2 is a member of the
immunoglobulin superfamily expressed on endothelium and on certain
leukocyte subpopulations.41 In our hands ICAM-2 expression
was detected only in patent vasculature (Figures 1G-1H'). Quantitative imaging of the ICAM-2 staining showed that hemizygous and
null cultures had 50% and 10% of the vascular structures of wild-type
cultures, respectively (Figure 2A). The vasculature that formed in the
null mutant cultures was predominantly small and rounded, with a
paucity of branch points (Figure 1C).
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| Fig 1.
Immunofluorescent antibody staining with vascular markers
in VEGF mutant ES cell cultures.
ES cells were differentiated, fixed on day 6 (G, G'), day 8 (A-F,
I), or day 9 (H-H'), and stained with antibodies to ICAM-2 (A-C,
G', H') or PECAM (D-G, H, I), followed by a PE-conjugated
secondary antibody. (A, D, G, G', H, H'),VEGF +/+ cultures;
(B, E) VEGF +/ cultures; (C, F) VEGF /cultures; (I)
/VEGF culture differentiated in the presence of 15 ng/mL
recombinant hVEGF-165 from day 3 to day 8. The insets in D and F
illustrate the differences in PECAM staining patterns, with arrowheads
showing staining of borders between PECAM-expressing cells and arrows
showing staining of borders between PECAM-expressing and nonexpressing
cells. (G, G') +/+ day 6 culture stained with PECAM (G) and
ICAM-2 (G') to show the lack of ICAM-2 staining before the
development of patent vasculature. (H, H') +/+ day 9 culture
stained with PECAM (H) and ICAM-2 (H') to show ICAM-2 staining of
patent vasculature. Magnification, 50×.
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| Fig 2.
Quantitative imaging analysis of VEGF mutant ES cell
cultures.
Day 8 ES cells were processed as shown in Figure 1, and
imaging analysis was performed as described in "Materials and
methods." (A) Percentage area stained with antibody to ICAM-2 in 2 separate experiments. W, wild-type (+/+) VEGF cultures; H, hemizygous
(+/) VEGF cultures; N, null (/) VEGF cultures. In all
cases, (+) after the letter denotes the same genotype ES cells
incubated with 15 ng/mL recombinant hVEGF-165 from days 3 to 8. (B)
Percentage area stained with antibody to PECAM or ICAM-2 in duplicate
wells of the same experiment. Abbreviations are as for A. In all cases,
statistical analysis was performed comparing H and N figures to the W
figure in the same experiment, and each (+) figure was compared to the
nonrescued figure for the same genotype. *P < .01;
**P < .001. (C) Semiquantitative assessment of PECAM+
aggregates (examples in Figures 1F, 1G) was conducted by visual
inspection of 2 to 4 wells of each genotype in separate
experiments. The relative abundance of the aggregate staining was
estimated as follows: +/, trace; 1+, 2% to 5%; 2+, 5% to 10%;
3+, 10% to 30%, 4+, more than 30%.
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The staining pattern of another vascular cell adhesion molecule, PECAM,
indicated a similar reduction in the amount of patent vasculature in
the mutant cultures (Figures 1D-1F). However, a second population of
PECAM+ cells was identified that formed small clumps that were not
patent vasculature (Figure 1F). These clumps of PECAM+ cells were rare
in wild-type cultures, more prevalent in hemizygous cultures, and
fairly abundant in null cultures (Figure 2C). In the cell clumps at
least part of each cell border was free of PECAM antibody staining,
whereas in the patent vasculature the PECAM staining pattern was
continuous for each endothelial cell (Figure 1, insets to D and F).
Moreover, the PECAM+ cells in the clumps appeared more rounded than the
endothelial cells incorporated into vascular structures. The staining
pattern seen in the clumps closely resembled the pattern of PECAM
staining seen at early times during ES cell differentiation before the development of vascular structures (Figure 1G and 12).
Quantitative imaging of wells stained with PECAM showed that the
PECAM-stained area was increased in hemizygous and null cultures
compared with wild-type cultures, though only the increase in
hemizygous cultures was statistically significant (Figure 2B). This
contrasted with the levels of ICAM-2 staining and was consistent with
the observation that increased numbers of PECAM+ cells were found in
clumps as the number of PECAM+ and ICAM-2+ vascular structures decreased.
The addition of exogenous recombinant VEGF (human 165 isoform)
from day 3 to day 8 of differentiation enabled the rescue of vascular
development in the null mutant cultures (Figures 1I and 2A). ICAM-2
staining of rescued null mutant cultures hemizygous showed amounts of
patent vasculature similar to those of the hemizygous cultures.
However, in no case were mutant cultures rescued to wild-type levels of
vasculature (Figure 2A). Moreover, the exogenous VEGF had little effect
on the hemizygous cultures and no significant effect on the wild-type
cultures. Optimal rescue was seen at 15 ng/mL, with lower
concentrations giving less rescue and higher concentrations showing no
significant differences from 15 ng/mL effects (data not shown). These
findings showed that the soluble human VEGF 165 isoform could rescue
vascular development in the cultures, but they suggested that
additional components were required to bring vascular development to
wild-type levels.
RNA analysis shows effects of the VEGF mutation
To further characterize the effects of the lack of VEGF signaling
during ES cell differentiation, RNase protection analysis was performed
on day 8 cultures (Figures 3,
4). For each reaction an antisense probe to
-actin was included and was used to normalize the radioactivity in
the protected bands. The levels of VEGF RNA showed the expected
relationships among the cultures (Figures 3A, 4),21 and
they mirrored the relative amounts of vasculature defined by ICAM-2
staining (Figure 2). The targeted VEGF locus produces a small amount of
truncated transcript that does not encode a functional
protein,21 and Northern blot analysis showed only the
truncated transcript in RNA from the null VEGF mutant cultures (data
not shown). The absence of functional VEGF in the null mutant cultures
indicated that the residual vascular development must have resulted
from some other mechanism.
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| Fig 3.
RNA protection analysis of VEGF mutant ES cell cultures.
Total RNA from cultures on day 8 of differentiation was hybridized with
the indicated 32P-labeled probes and a -actin probe as
described in "Materials and methods." After separation on a
polyacrylamide-urea gel, protected fragments were visualized and
quantitated using a PhosphorImager. Lane 1, VEGF (+/+) ES culture RNA;
lane 2, VEGF (+/) ES culture RNA; lane 3, VEGF (/) ES
culture RNA; lane 4, NIH 3T3 fibroblast cell line RNA; lane 5, Py-4-1
endothelial cell line RNA. (A) Protection with antisense VEGF probe.
(B) Protection with antisense PECAM probe. (C) Protection with
antisense flt-1 probe. (D) Protection with antisense tie-2/tek probe.
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| Fig 4.
Quantitative RNA analysis of VEGF mutant ES cell
cultures.
PhosphorImager data from the gels in Figure 3 were manipulated to
subtract background and to normalize to the -actin protected signal
in the same lane. Relative band densities for each probe in het (+/)
and null (/) lanes were then compared to (+/+) wild-type
levels for that probe. Each result, except for the VEGF analysis, is a
compilation of at least 3 experiments on at least 2 (and sometimes 3)
different sets of RNA prepared on different days and at different
passage numbers. In all cases, statistical analyses compared VEGF het
and null RNA levels to the VEGF wild-type RNA levels for that probe.
*P < .05; **P < .000 0001.
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RNA levels of the endothelial adhesion receptors ICAM-2 and PECAM also
showed the expected trends, though several differences between
relative RNA levels and quantitative imaging comparisons were noted
(Figures 2B, 4). ICAM-2 RNA levels were not significantly lower in
hemizygous than in wild-type cultures, but ICAM-2 protein imaging
analysis showed a 50% reduction in stained areas of the hemizygous
cultures compared with wild-type cultures (Figure 2B). Similarly, the
null cultures showed a significant decrease in ICAM-2 RNA levels but
only to 50% of the wild-type and hemizygous levels, suggesting that
ICAM-2 RNA may accumulate in the absence of translation to protein.
Levels of PECAM RNA decreased to 75% of wild-type levels in hemizygous
cultures but were not further decreased in null mutant cultures.
Overall the trends for each adhesion receptor were similar between
imaging and RNA analyses, and they were consistent with the presence of
nonvessel PECAM+ cells in the mutant cultures.
RNA levels for the high-affinity VEGF receptors showed
different trends in response to the lack of VEGF signaling
(Figures 3, 4). Levels of flk-1 RNA did not vary significantly among
the different cultures, but the amount of flt-1 RNA in the null mutant VEGF cultures decreased to half the levels seen in the wild-type and
hemizygous cultures. These data show that RNA for the 2 receptors accumulated differently in the absence of VEGF signaling. A third endothelial tyrosine kinase receptor not involved in VEGF
signaling, tie-2/tek, did not show significant changes in RNA
levels among the different cultures.
Characterization of PECAM+ cells not incorporated into
vasculature
Double-label immunofluorescent antibody staining was performed on
day 8 VEGF mutant cultures to further characterize the PECAM+ cells
found in clumps. As expected from the initial staining, double labeling
with ICAM-2 and PECAM showed that the PECAM+ cell clumps did not
express detectable ICAM-2 (Figure 5).
Moreover, the PECAM+, ICAM-2 clumps were more prevalent in
hemizygous cultures than in wild-type cultures and more prevalent still
in null VEGF mutant cultures. This staining pattern confirmed
that only vascular endothelial cells incorporated into patent
vasculature expressed detectable ICAM-2. A second marker of early
vasculature, CD34, was also not detectable in the PECAM+ clumps (Figure
6). CD34 staining, in fact, mirrored ICAM-2
staining, and only vascular endothelial cells that were incorporated
into patent vasculature expressed both CD34 and ICAM-2. However, some
CD34+ cells were found outside the vasculature and did not express
either PECAM or ICAM-2 (Figure 6). In contrast, ICAM-2 staining
was confined to the patent vasculature and possibly to hematopoietic
cells within the blood islands.
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| Fig 5.
PECAM/ICAM-2 double-label immunofluorescent antibody
staining of VEGF mutant ES cultures.
Day 8 cultures were fixed and stained with rat antimouse ICAM-2 (red)
and rabbit polyclonal antimouse PECAM (green) antibodies, along with
the appropriate secondary antibodies. Images were photographed with the
appropriate FITC (B, E, H) or rhodamine (C, F, I) filters or with a
double exposure using both filters sequentially (A, D, G). (A-C)
Wild-type (+/+) VEGF ES cultures. (D-F) Hemizygous (+/) VEGF mutant
ES cell cultures. (G-I) Null (/) VEGF mutant ES cultures.
The arrows in (D-I) point to clumps of cells that stain with the PECAM
antibody but not with the ICAM-2 antibody. Magnification,
50×.
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| Fig 6.
PECAM/CD34 double-label immunofluorescent antibody
staining of hemizygous VEGF mutant ES cultures.
Day 8 VEGF hemizygous (+/) ES cultures were fixed and stained with
rat antimouse CD34 (red) and rabbit polyclonal antimouse PECAM (green)
antibodies, along with the appropriate secondary antibodies. Images
were photographed with the appropriate FITC (B) or rhodamine (C)
filters or with a double exposure using both filters sequentially (A).
The arrow in each frame points to a clump of cells that stained with
the PECAM antibody but not with the CD34 antibody. The arrowheads in
each frame point to several individual cells that stained with the CD34
antibody but not with the PECAM antibody. Magnification, 50×.
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Double-label immunofluorescent antibody staining with
PECAM and SSEA-1 (Figure 7)
showed that some of the PECAM+ cells in clumps also stained for the
SSEA-1 marker. In contrast, none of the patent vasculature stained with
SSEA-1. SSEA-1 reacts with a carbohydrate epitope that is expressed on
undifferentiated and partially differentiated embryonic
cells.42,43 Although many of the small PECAM+ clumps
found in the wild-type cultures appeared to contain only
double-positive cells (Figure 7A), the larger clumps in
hemizygous and null cultures seemed to have both double-positive cells
and cells that stained only for PECAM (Figures 7D, 7G).
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| Fig 7.
PECAM/SSEA-1 double-label immunofluorescent antibody
staining of VEGF mutant ES cultures.
Day 8 cultures were fixed and stained with mouse antimouse SSEA-1 (red)
and rabbit polyclonal antimouse PECAM (green) antibodies, along with
the appropriate secondary antibodies. Images were photographed with the
appropriate FITC (B, E, H) or rhodamine (C, F, I) filters or with a
double exposure using both filters sequentially (A, D, G). (A-C)
Wild-type (+/+) VEGF ES cultures. (D-F) Hemizygous (+/) VEGF mutant
ES cultures. (G-I) Null (/) VEGF mutant ES cultures.
Magnification, 50×.
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Expression of flk-1 in vascular endothelial growth factor mutant
cultures
Because the amount of flk-1 RNA did not decrease in the
mutant cultures, though the amount of vasculature was dramatically reduced, we examined the localization of flk-1 RNA-expressing cells by
in situ hybridization analysis and compared the flk-1 pattern to the
PECAM pattern (Figure 8). As expected, in
wild-type cultures both probes were localized to patent
vasculature (Figures 8A, 8B). In hemizygous cultures hybridized with
the PECAM probe, patent vasculature and clumps of PECAM+ cells were
seen (Figure 8C). The hemizygous cultures hybridized with the flk-1
probe showed patent vasculature, small clumps of cells, and isolated
single cells that stained (Figure 8D). The null cultures showed
different but partially overlapping patterns of expression for the 2 probes. The PECAM probe hybridized to numerous clumps of cells in
the null cultures that resembled in size and morphology the PECAM+ cell
clumps stained with the antibody (Figure 8E, 8G). The PECAM probe also
hybridized to the small amount of patent vasculature present (data not
shown). The flk-1 probe hybridized to areas of loosely aggregated
mesenchymal-looking cells (Figure 8F and to the small amount of patent
vasculature and clumps of cells that resembled the PECAM+ cells (Figure
8H).
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| Fig 8.
RNA in situ hybridization of VEGF mutant ES cultures.
Day 8 cultures were fixed and processed for in situ hybridization as
described in "Materials and methods." Cultures were hybridized
with an antisense PECAM probe (A, C, E, G) or an antisense flk-1 probe
(B, D, F, H). (A, B) Wild-type (+/+) VEGF ES cultures. (C, D)
Hemizygous (+/) VEGF mutant ES cultures. (E-H) Null
(/) VEGF mutant ES cultures. Magnification, (A-F)
50×, (G-H) 100×.
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To determine whether mutant cells expressing both PECAM
and flk-1 were present in the VEGF null cultures, double-label
immunofluorescence with PECAM and flk-1 antibodies was performed
(Figure 9). VEGF null cultures at day 5 and
day 6 of differentiation had cells that stained for both PECAM and
flk-1 and cells that stained only for PECAM. Some of the
double-positive cells were incorporated into patent vasculature, and
some of the double-positive cells were found in the PECAM+ clumps of
cells (Figure 9A-9C). However, not all cells of the PECAM+ clumps had
detectable flk-1 staining, and some small clumps of PECAM+ cells had no
detectable flk-1 staining (Figures 9D-9F). Similar staining patterns
were seen on days 7 and 8, but the intensity of the flk-1 antibody
signal was significantly reduced (data not shown). Interestingly, few cells had detectable flk-1 antibody staining but no detectable PECAM
antibody staining (data not shown), suggesting that flk-1 RNA may be
expressed in the mutant cells in the absence of detectable levels of
flk-1 protein.
View larger version (67K):
[in this window]
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| Fig 9.
PECAM/flk-1 double-label immunofluorescent antibody
staining of VEGF mutant ES cultures.
Day 5 (A-C) or day 6 (D-F) null (/) VEGF mutant cultures
were fixed and stained with rat antimouse PECAM (green) and rabbit
antimouse flk-1 (red) antibodies, along with the appropriate secondary
antibodies. Images were photographed with the appropriate FITC (C, F)
or rhodamine (B, E) filters or with a double exposure using both
filters sequentially (A, D). The arrows in A to C point to a clump of
PECAM+ cells that partially stained for flk-1. The structure in the
upper left is patent vasculature. Arrowheads in D to F point to clumps
of PECAM+ cells that had no detectable staining for flk-1. The
structure to the left is patent vasculature. Magnification, 50×.
|
|
| |
Discussion |
The role of the VEGF signaling pathway in vascular development was
examined by differentiating and analyzing ES cells with a targeted
mutation at the VEGF locus. Our results clarify that VEGF signaling is
crucial to vascular development, and they provide quantitative
information about the effects of both the hemizygous and homozygous
VEGF mutation on vasculogenesis. Most important, a population of
precursor cells that exhibit a unique marker pattern accumulates in the
mutant cultures. The reciprocal relationship between the amount of
patent vasculature and the precursor cells suggests that they are
blocked from further development by the absence of VEGF signaling
during ES cell differentiation. This is the most specific
understanding to date of the first point at which VEGF signaling is
critical to embryonic blood vessel formation.
Our quantitation of the effects of the VEGF mutation showed that
vascular development was reduced by approximately 50% in the
hemizygous VEGF mutant cultures and that VEGF RNA levels were also
reduced by approximately 50% in these cultures. Previous analysis of
the requirement for VEGF in vivo revealed that the loss of 1 copy of
VEGF produced embryonic lethality, suggesting a tight dose requirement
for VEGF signaling during vascular development.21,22 Although the null mutant VEGF cultures did not produce functional VEGF
protein, a low but reproducible level of vascular development did
occur, as defined by the presence of groups of cells with contiguous
cell border expression of PECAM and ICAM-2. This primitive blood vessel
formation occurred at levels reduced by approximately 10 times those in
wild-type cultures. This finding suggests that blood vessel formation
is not completely dependent on signaling mediated by VEGF (VEGF-A), and
it is consistent with the presence of some vasculature in the VEGF null
mutant embryos.21 The recent identification of several
VEGF-related molecules indicates that some redundancy may exist in the
VEGF signaling required for vascular development. For example, both
VEGF-C and VEGF-D can signal through the flk-1 (VEGFR-2) receptor,
whereas PIGF and VEGF-B can signal through the flt-1 (VEGFR-1)
receptor. Moreover, a third receptor, called flt-4 (VEGFR-3), can
signal through VEGF-C and VEGF-D,25 and a targeted mutation
in flt-4 is a homozygous embryonic lethal with vascular
defects.44 In fact, in light of the plethora of VEGF-related molecules and receptors, it seems surprising that the VEGF
mutation has such a profound effect on embryonic vascular development
because our study clearly showed that 90% of vascular development
during ES cell differentiation required VEGF-dependent signaling.
VEGF signaling is also implicated in the expansion and branching of
endothelial cells that occur after primary differentiation events, and
our results indicate that VEGF is important for these events during ES
cell differentiation. The vessels that formed in the VEGF mutant
cultures were small and rounded, and they had fewer branch points than
wild-type cultures when normalized to the amount of vasculature (Scalia
A, Bautch VL, unpublished results). Vascular defects were partially
rescued by addition of exogenous VEGF-165, which produced more
vasculature with more branch points. Thus this isoform is likely to
make major contributions to VEGF signaling in vivo, consistent with in
vitro studies showing that VEGF-165 has strong mitogenic
and chemotactic activities. Our inability to rescue vascular
development completely with exogenous VEGF-165 indicates that either
the local concentration or presentation of exogenous VEGF did not reach
levels sufficient for complete rescue or that other isoforms also
contribute significantly to VEGF signaling in vivo.
Quantitative RNA analysis showed that the 2 high-affinity VEGF
receptors, flk-1 and flt-1, responded differently to the absence of
ligand. The flk-1 levels did not differ significantly among the
genotypes, and the lack of patent vasculature in the mutant cultures
suggested that flk-1 RNA expressing precursor cells accumulated in the
absence of VEGF signaling. This was confirmed by in situ hybridization
analysis that showed strong flk-1 RNA expression from dispersed cells
and clumps of cells. Thus flk-1 RNA expression is activated in the
absence of VEGF, but the flk-1 expressing cells do not efficiently
mature to endothelial cells that form patent vasculature.
Interestingly, the number of cells expressing detectable flk-1 protein
were reduced in the VEGF null mutant cultures compared with the flk-1
RNA expression pattern, suggesting that in the absence of VEGF the
receptor protein is either not translated at high levels or is not
stable. In contrast, flt-1 RNA levels were maintained in
hemizygous VEGF mutant cultures but decreased significantly in null
VEGF mutant cultures. The VEGF mutant cultures may express flt-1 RNA at
reduced levels because VEGF signaling is required to regulate
expression of the flt-1 receptor.45
The trends seen in the relative RNA levels of the 2 vascular adhesion
receptors, ICAM-2 and PECAM, in general followed the patterns seen with
the quantitative imaging of the staining of each protein. In our hands
the ICAM-2 antibody reacted only with endothelial cells incorporated
into patent vasculature, and ICAM-2 RNA levels and antibody-stained
areas decreased with reduced VEGF production. In contrast, PECAM
antibody stained clumps of unincorporated cells and patent vasculature,
and both the quantitative imaging and the relative RNA levels reflect
this expanded PECAM expression profile relative to ICAM-2. These
results show that the quantitative change in PECAM expression levels
was minimal, yet the corresponding immunofluorescent antibody staining
patterns show a dramatic difference in the type of cells stained in the
mutant series.
The PECAM+ cells that accumulated in the VEGF mutant cultures
may be precursor cells blocked from further differentiation by the lack
of VEGF signaling. Several lines of evidence support this hypothesis.
First, there is a rough reciprocal relationship between the amount of
patent vasculature and the amount of PECAM staining outside vessels.
Wild-type cultures have only rare clumps of PECAM+ cells, hemizygous
cultures have fewer patent vessels and more PECAM+ clumps, and null
cultures have only rare small vessels and numerous clumps of PECAM+
cells. Second, similar clumps of PECAM+ cells are prevalent at early
times of in vitro differentiation, before the development of patent
vasculature.12 Third, clumps of mesodermal cells that
aggregate in the yolk sac just before blood island formation express
PECAM and resemble in morphology the PECAM+ cell clumps seen during ES
cell differentiation.10,12 Finally, rescue of the VEGF null
cultures by exogenous VEGF produces more patent vasculature and
correspondingly fewer clumps of PECAM+ cells, suggesting that VEGF can
promote the maturation of the PECAM+ cell clumps into patent vasculature.
Several markers distinguish the PECAM+ cells in clumps from those in
vessels. ICAM-2 and CD34 are both expressed by endothelial cells
incorporated into patent vasculature but not by PECAM+ cell clumps.
This marker profile also distinguishes the PECAM+ clumps in normal ES
cell cultures that form early in differentiation from the patent
vasculature that forms later.12 CD34 is expressed on early
vessels and hematopoietic cells in mouse development,46 but
it has not been associated with immature endothelial cells; in a recent
study, it was also associated with more mature ES cell-derived
endothelial cells.7 This study also identified VE-cadherin
as an early marker of the endothelial lineage, so it will be
informative to determine the expression of VE-cadherin relative to
PECAM in the VEGF mutant cultures. The developmental expression of
ICAM-2 is less well documented. ICAM-2 RNA is detected in a vascular
and endocardial pattern during mouse embryogenesis, but ICAM-2 RNA
expression is not associated with early yolk sac mesodermal
condensations (Bautch VL, unpublished results).
In contrast, SSEA-1 is expressed by a subset of the PECAM+ cells in
clumps but not by endothelial cells incorporated into vessels.
SSEA-1 recognizes a carbohydrate epitope that is expressed by several
embryonic lineages, so it is less useful as a lineage marker than as a
way to distinguish PECAM+ cell clumps from PECAM+ vasculature. Flk-1
protein is also detectable on a subset of PECAM+ cells in clumps, which
is consistent with a model in which PECAM+ cells in clumps respond to
VEGF signaling through the flk-1 receptor to mature into endothelial
cells. Flk-1 is also expressed on patent vasculature early during ES
cell differentiation. Our results are consistent with dual roles for
VEGF/flk-1 receptor signaling in endothelial cell differentiation and
in expansion and branching of endothelial cells. A recent study
analyzed flk-1 null mutant ES cell differentiation and suggested that
flk-1 signaling was not required for endothelial cell
differentiation.36 However, in our hands, ES cells carrying
a homozygous null mutation for flk-1 differentiated similarly to the
VEGF null mutant ES cells, with an accumulation of PECAM+ cell clumps
and residual patent vasculature that was positive for PECAM and
ICAM-2 (Lau DC, Bautch VL, unpublished results). Thus our data support
a model that has a primary requirement for VEGF/flk-1 receptor
signaling in endothelial cell differentiation that is not absolute and
a requirement for the same signaling pathway in later stages of
vascular development, such as endothelial cell expansion and branching.
| |
Acknowledgments |
We thank Dan Dumont and Kevin Peters for the probes and Beat Imhof
for the polyclonal PECAM antiserum. We thank Susan Whitfield and David
Miller for artwork. We thank fellow laboratory members for fruitful
discussions. The MC-480 hybridoma was obtained from the Developmental
Studies Hybridoma Bank maintained by the Department of Pharmacology and
Molecular Sciences, Johns Hopkins University School of Medicine
(Baltimore, MD) and the Department of Biological Sciences,
University of Iowa, (Iowa City, IA) under contract N01-HD-6-2915 from
the NICHD.
| |
Footnotes |
Submitted July 12, 1999; accepted November 3, 1999.
Supported by National Institutes of Health grant HL43174 to V.L.B. and
by grants from the European Community (Biomed BMH4-CT98-3380) and Actie
Levenslijn (#7.0019.98) to P.C. V.L.B. was supported by a National
Institutes of Health RCDA Award, and S.D.R. was supported by a National
Institutes of Health NSRA Award.
Reprints: Victoria L. Bautch, Department of Biology, University
of North Carolina at Chapel Hill, CB#3280, Chapel Hill, NC 27599;
e-mail: bautch{at}med.unc.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Top
Abstract
Introduction