Alternative Splicing of Vascular Endothelial Growth Factor (VEGF)-R1 (FLT-1) pre-mRNA Is Important for the Regulation of VEGF Activity
Yulong He,
Stephen K. Smith,
Kate A. Day,
Dawn E. Clark,
Diana R. Licence and
D. Stephen Charnock-Jones
Reproductive Molecular Research Group Department of Obstetrics
and Gynaecology University of Cambridge The Rosie Hospital
Cambridge, CB2 2SW, UK
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ABSTRACT
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Angiogenesis is essential for normal
mammalian development and is controlled by the local balance of pro-
and antiangiogenic factors. Here we describe a novel mouse cDNA
sequence encoding sFLT-1 that is a potent antagonist to vascular
endothelial growth factor (VEGF) and show for the first time its
in vivo production. In situ hybridization and
Northern blot analysis with probes specific for sFLT-1 or FLT-1 showed
that the relative abundance of their mRNAs changed markedly in
spongiotrophoblast cells in the placenta as gestation progressed. On
day 11 of pregnancy, sFLT-1 mRNA was undetectable but FLT-1 readily
apparent, and by day 17 sFLT-1 mRNA was abundant but FLT-1 barely
detectable. sFLT-1 was identified in conditioned medium of cultured
placenta from day 17 pregnant mice and likely to be present in the
circulation, as there is a substantial increase of VEGF-binding
activity in the serum from day 13 of pregnancy, which coincides with
the abundant sFLT-1 expression in placenta. Expression of sFLT-1 was
also observed in adult lung, kidney, liver, and uterus. These data
suggest a novel mechanism of regulation of angiogenesis by alternative
splicing of FLT-1 pre-mRNA. Treatment of pregnant mice with exogenous
VEGF from day 9 to 17 of pregnancy, which alters the ratio of VEGF to
sFLT-1, resulted in an increase in the number of resorption sites and
fibrin deposition in the placenta of ongoing pregnancies. These
findings have important implications for understanding placental
function and may be relevant in a range of disease states.
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INTRODUCTION
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Angiogenesis, the sprouting of new capillaries, is rare in
the adult with exceptions in the female reproductive tract and in
pathological conditions (1, 2, 3). Mammalian placentation requires
extensive angiogenesis to establish a suitable vascular network for the
supply of oxygen and nutrients to the fetus (4). A variety of
angiogenic growth factors including fibroblast growth factors
(5, 6, 7) and some of the vascular endothelial growth factor (VEGF) family
(8, 9, 10, 11, 12) are expressed in placenta. However, rapid growth of placenta in
early pregnancy is accomplished in an unusually hypoxic environment
(13), suggesting that hypoxia-induced angiogenesis mediated by
angiogenic activators such as VEGF and its receptors may be of
particular importance (14, 15). In the placenta, VEGF transcripts were
detected in maternal and labyrinthine layers and also in trophoblast
giant cells (10, 11, 16). VEGFR1 (FLT-1) is found in the
spongiotrophoblast layer and VEGFR2 (FLK) in labyrinthine layer (10, 11). In tissues where blood vessel growth is occurring, the net
angiogenic effect is controlled by the balance between angiogenic
inducers and inhibitors (17). Thus the identification of specific
antiangiogenic agents in the placenta, such as PRP (18) and sFLT-1,
described in this paper, is of considerable importance for
understanding placental and fetal growth.
Vascular endothelial growth factor (VEGF), a potent and
endothelial-specific mitogen, has been demonstrated to have a pivotal
role in vasculogenesis and angiogenesis (19, 20, 21, 22). This protein was
independently isolated as vascular permeability factor (23) and is a
potent stimulator of this process (24, 25). Alternative splicing of the
pre-mRNA encoding one of the VEGF receptors (FLT-1) results in the
production of a soluble form comprising the ligand-binding domain
of this receptor (sFLT-1), which is a potent antagonist of VEGF (26).
In addition to inhibiting VEGF binding to cell surface receptors,
sFLT-1 also forms heterodimers with the other VEGF receptor, KDR (27).
This would account for the efficient inhibition of VEGF function by
sFLT-1. Soluble FLT-1 chimeric proteins have also been shown to
suppress retinal neovascularization (28) and corpus luteum angiogenesis
(29). However, until now there are no data available showing whether
sFLT-1 is present in vivo, or when and where it may be
produced. We and others have previously shown the expression of FLT-1
by human trophoblast (30, 31) and mouse spongiotrophoblast (10, 11). In
this study we cloned a 3'-end cDNA fragment of mouse sFLT-1 and showed
stage-dependent expression of FLT-1 and sFLT-1 in placenta, suggesting
a novel mechanism of regulation of angiogenesis by alternative splicing
of FLT-1 pre-mRNA. We also explored the effect of exogenous VEGF on the
developing placenta in mice, which showed that perturbation of the VEGF
to sFLT-1 ratio by administration of exogenous VEGF led to fibrin
deposition in the placenta and a reduction in embryo weight.
Angiogenesis is currently the target for therapies in a wide range of
diseases, including cancer, retinopathy, and heart disease. These
findings have important implications for the regulation of angiogenesis
in mammals.
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RESULTS
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Cloning of 3'-End of Mouse sFLT-1
The 3'-end of mouse sFLT-1 was cloned using 3'-RACE (rapid
amplification of cDNA ends) and characterized by DNA sequencing. The
cloned fragment includes 150 bp of coding sequence, an inframe stop
codon, and 18 bp of noncoding sequence followed by a poly (A)+ tail
(EMBL accession No. AJ001177). This 171-bp fragment shares 85.5%
nucleotide sequence identity to the corresponding region of human
sFLT-1 as determined by the alignment program GAP (GCG, Madison,
WI). However, the 3'-noncoding sequence of mouse sFLT-1 is much shorter
than that of human sFLT-1 (338 bp) (26). The nucleotides around the
site of FLT-1 and sFLT-1 divergence are identical in human and mouse,
suggesting that splice site skipping occurs in the mouse as well as in
the human, as suggested by Kendall and Thomas (26). Alignment of the
3'-end cDNA (which is not present in FLT-1) and the deduced C-terminal
amino acid sequence of mouse sFLT-1 with those of human sFLT-1 is shown
in Fig. 1
.

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Figure 1. Alignment of the 3'-End of cDNA and C-Terminal
Amino Acid Sequence of Mouse sFLT-1 (m) with Those of Human sFLT-1 (h)
The deduced C-terminal amino acid sequence of mouse and human sFLT-1
are above or below their cDNA sequence,
respectively. Arrowhead, Divergent site of
membrane-bound FLT-1 and sFLT-1.
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sFLT-1 Expression Increased, but FLT-1 Decreased, in Placenta in
Late Gestation
To investigate the sites and relative expression levels of FLT-1
and sFLT-1 in the placenta, in situ hybridization using
probes specific for the two forms was performed on sections prepared
from placentas at different stages of gestation. The specificity of the
probes demonstrated by Northern blot is shown in Fig. 2
. sFLT-1 transcripts were detected in
the placental spongiotrophoblast cells of days 13 (Fig. 3E
), 15 (Fig. 3H
), and 17 (Fig. 3I
). No
hybridization signals were observed on day 11. Serial sections were
also hybridized with the antisense FLT-1 probe. FLT-1 expression was
observed in the spongiotrophoblast cells of days 11 (Fig. 3A
), 13 (Fig. 3D
), and 15 (Fig. 3G
), but only weak signals were found on day 17.
Serial sections hybridized with the sense sFLT-1 (Fig. 3B
) and FLT-1
(data not shown) probes did not show signal above the background.

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Figure 2. Northern Blot Demonstrating the Specificity of the
Probes for FLT-1 (A), or sFLT-1 (B) Transcripts
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Figure 3. In Situ Hybridization Analysis of
FLT-1 and sFLT-1 Expression in Placenta at Different Stages of
Pregnancy
At each stage, serial sections of the same placenta were used for a
comparative analysis using specific FLT-1 and sFLT-1 probes. FLT-1
expression was detected in the spongiotrophoblast cells of days 11 (A),
13 (D), and 15 (G). sFLT-1 transcripts were localized in the
spongiotrophoblast cells of days 13 (E), 15 (H), and 17 (I). F, Bright
field image of panel I; C, higher magnification of panel F. B,
Hybridization of serial sections with sense sFLT-1 probe did not
produce any signal above the background. la, Labyrinthine layer;
arrowhead, spongiotrophoblast layer; m, maternal layer.
White scale bar, 200 µm (A, B, D, E, F, G, H, and I);
black scale bar, 60 µm (C).
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Total RNA from days 13, 15, or 17 placenta was also analyzed by
Northern blot using a cDNA probe detecting both sFLT-1 and FLT-1
transcripts, and two bands were detected in each sample (Fig. 4
). The level of sFLT-1 transcripts was
much higher than that of FLT-1, and the ratio of sFLT-1 to FLT-1
changed markedly as gestation progressed. This result is consistent
with the observation from in situ hybridization.

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Figure 4. Northern Blot Analysis of Total RNAs from Placenta
of Days 13 (lane 1), 15 (lane 2), and 17 (lane 3) Using a cDNA Probe
for Detecting Both FLT-1 and sFLT-1 Transcripts
Upper arrowhead, FLT-1 transcripts; lower
arrowhead, sFLT-1 transcripts.
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sFLT-1 Is Also Expressed in Adult Organs
Sections from adult lung, liver, kidney, and uterus were also
analyzed by in situ hybridization, but no signals above
background were detected. However, RT-PCR for 28 cycles using specific
primers demonstrated that sFLT-1 transcripts could be detected in adult
kidney, lung, and uterus (Fig. 5
) and
also in liver if 35 cycles were used (data not shown). The same amount
of RNA without RT was used as control and no band was detected.

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Figure 5. RT-PCR to Specifically Detect sFLT-1 mRNA in
Different Adult Tissues
Lane 1, kidney; lane 2, liver; lane 3, lung; lane 4, uterus; lane 5,
placenta; lane 6, negative control.
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Characterization of the Placental VEGF-Binding Factor
Serum-free conditioned medium of mouse placenta contains
VEGF-binding activity. To determine whether this VEGF-binding activity
is related to sFLT-1, a fraction of heparin-Sepharose partially
purified protein from placenta- conditioned medium was further analyzed
by Western blot. A protein band, approximately 111 kDa, was detected by
antihuman FLT-1 antibody (Fig. 6
, lane 2)
and also by anti-FLT-1 N-terminal antibody (data not shown). This is in
good agreement with Kendall et al. (27), who found that
endothelial cells produced a sFLT-1 of 110 kDa. These authors (26) also
showed that baculovirus-expressed sFLT-1 had an approximate molecular
mass of 90 kDa. This is consistent with our results (Fig. 6
, lane 1). The size difference between the endogenously encoded and
recombinant sFLT-1 is probably due to the differential glycosylation of
protein in mammalian and insect cells. In control immunoblots, rabbit
IgG instead of FLT-1 antibody was used, and no band was detected (data
not shown).

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Figure 6. The VEGF-Binding Activity from Conditioned Medium
of Mouse Placenta (lane 2) and Baculovirus-Expressed Human Recombinant
sFLT-1 (lane 1) Were Separated by 412% Bis-Tris NuPAGE and Analyzed
by a Western Blot Probed with the Biotinylated anti-Human FLT-1
Antibody
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The VEGF-Binding Activity Increases in the Serum of Mice in Late
Gestation
Since sFLT-1 transcripts became much more abundant as gestation
progressed, we investigated whether sFLT-1 protein was detectable in
the maternal circulation at this time. After incubation of serum with
[125I]VEGF, complexes between [125I]VEGF
and the VEGF-binding activity were formed and separated using a
Sephacryl S-200 gel filtration column. Fractions eluted in PBS were
collected and the radioactivity in each was determined. Serum from
nonpregnant and day 11 pregnant mice produced a similar pattern of
peaks (Fig. 7
), with one small peak at
fraction 10 and two additional peaks around fractions 14 and 28.
However, serum from days 13, 15, or 17 pregnant mice showed a different
peak pattern, with a major peak at fraction 10 (Fig. 7
). The peak
around fraction 28 remained in all samples. When 100-fold excess of
unlabeled recombinant human VEGF was coincubated with serum from day 17
pregnant mice and [125I]VEGF, the major peak at fraction
10 disappeared, indicating specific binding between VEGF and the
VEGF-binding activity. When serum from a nonpregnant mouse, to which
recombinant human sFLT-1 had been added, was coincubated with
[125I]VEGF, a major peak appeared at fraction 10,
indicating the chromatographic similarity between recombinant sFLT-1
and the serum VEGF-binding factor. Incubation of
[125I]VEGF with PBS also produced two peaks, one at
fraction 14 and the other at 28. These correspond to the complexes of
[125I]VEGF and BSA and free [125I]VEGF.
[125I]VEGF is supplied with BSA carrier, and ligand blots
show VEGF interacts weakly with BSA (data not shown).

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Figure 7. Sephacryl S-200 Gel Filtration Chromatography
The peak at fraction 10 is the complexes of [125I]VEGF
and the VEGF-binding activity, and the peak around fraction 14 is
[125I]VEGF and BSA complexes. Free VEGF produces a peak
around fraction 28. np, Serum from nonpregnant mice.
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Administration of Exogenous VEGF Led to Fibrin Deposition in the
Placenta
The presence of a potent VEGF antagonist suggests that the action
of this growth factor is regulated. We investigated this by perturbing
the ratio of VEGF to sFLT-1 by administration of exogenous recombinant
VEGF to pregnant mice. In animals treated with recombinant VEGF, there
was an increase in the number of resorption sites. There were 18
resorption sites present in the treated mice (n = 5) and only 1 in
the control mice (n = 4) (P < 0.05, Wilcoxon rank
sum test). Embryos from these mice, which appeared macroscopically
normal, weighed less than those from the control group (mean ±
SD: control, 0.53 ± 0.05 g; treated, 0.46
± 0.09 g P < 0.001, Students t
test). There was no difference in the placental weight between these
two groups. However, histological examination of placenta from these
two groups did reveal differences. In the treated group there was an
increase in fibrin deposition apparent within the labyrinthine layer
(Fig. 8
). The deposition was widespread
as shown by the red/purple stain around many of the cells (Fig. 8
, A
and B). There were also occasional areas of intense staining (Fig. 8
, C
and D). Such staining was absent from the placentas of the
vehicle-treated mice (Fig. 8
, E and F).

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Figure 8. Histology of Mouse Placental Sections Stained with
MSB to Reveal Fibrin Deposition Obtained from Mice Treated with VEGF
(A, B, C, and D) or Vehicle (E and F) during Pregnancy
Fibrin deposits are bright red, red blood cells are
stained yellow, and other cells are stained
blue. Scale bar, 50 µm in panels A, C, and E; 10 µm
in panels B, D, and F.
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DISCUSSION
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In this study we have shown the stage-dependent expression of
FLT-1 and sFLT-1 in placental spongiotrophoblast during pregnancy in
the mouse. This suggests a novel mechanism for the regulation of VEGF
activity by alternative splicing of FLT-1 pre-mRNA.
The mRNA encoding full-length membrane-bound FLT-1 was detected in
placental spongiotrophoblast cells on day 11 of pregnancy, but sFLT-1
mRNA was undetectable by in situ hybridization at this time.
However, high levels of sFLT-1 transcripts were observed on day 13 and
rose as gestation progressed. In contrast, FLT-1 transcripts declined
in late gestation such that by day 17 they were almost undetectable by
in situ hybridization. This suggests that there may be
regulation of the splice site selection in these cells leading to a
marked shift in the ratio of their mature mRNAs. The change of the
sFLT-1 to FLT-1 ratio was further confirmed by Northern blot analysis
(Fig. 4
). sFLT-1 was identified in conditioned medium of cultured mouse
placenta. These results indicate that sFLT-1 is produced in the
placenta. Thus, it is likely that the VEGF-binding activity found in
the serum of mice in late pregnancy is sFLT-1. VEGF-binding activity
was also detected in the serum of pregnant women (data not shown),
suggesting that sFLT-1 may have a systemic role in antagonizing
increasing VEGF during pregnancy (32). Recent data have confirmed the
role of sFLT-1 as an inhibitor of angiogenesis. Intravitreal injection
of soluble VEGF-receptor chimeric proteins is able to suppress retinal
neovascularization in a murine model of ischemic retinopathy (28).
Furthermore, treatment of super-ovulated rats with truncated soluble
FLT-1 receptors resulted in the complete suppression of corpus luteal
angiogenesis and a failure of endometrial maturation (29). These
studies show that sFLT-1 is a potent antagonist of VEGF in
vivo. Thus, it is likely that the physiological alternative
splicing of FLT-1 pre-mRNA to generate FLT-1 and/or sFLT-1, described
in this paper, will be important for the regulation of placental
angiogenesis.
RT-PCR analysis showed that sFLT-1 mRNA is also present in adult lung,
liver, kidney, and uterus, suggesting that sFLT-1 may have a role in
maintaining endothelial cells in a quiescent state in the adult. sFLT-1
is also produced by human umbilical vein endothelial cells in
vitro (27). Thus, a similar alternative splicing mechanism for
FLT-1 pre-mRNA also exists in endothelial cells. Whether the
FLT-1/sFLT-1 switch is involved in the modulation of pathological
angiogenesis remains to be investigated.
It has been shown that the migration of monocytes/macrophages in
response to VEGF is mediated by FLT-1 (33, 34). Since FLT-1 mRNA was
detected in the trophoblast before day 13, it is possible that FLT-1
may regulate the migration of trophoblast cells.
The spongiotrophoblast cell layer where sFLT-1 transcripts are
localized is located between the maternal and labyrinthine layers where
VEGF is expressed (10, 11). The balance of the locally expressed VEGF
and sFLT-1 could be important in the regulation of placental
endothelial cell function. Administration of exogenous VEGF to pregnant
mice led to fibrin deposition in the placenta, an increase in
resorption sites, and a reduction in embryonic weight. This suggests
that the exogenous VEGF circumvents the regulatory control of the
angiogenic events in the placenta, and that a balance of angiogenic
inducers and inhibitors is critical for normal placental function. The
enhanced vascular permeability and the induction of tissue
factor synthesis by endothelium and monocytes in response to exogenous
VEGF (24, 25, 35) may contribute to fibrin deposition in the placenta
of VEGF-treated mice. The placental abnormalities may account for the
significant reduction of the embryo weight in these mice.
However, no abnormalities were observed in other organs of
VEGF-treated mice (data not shown). This may be a reflection of the
presence of locally acting inhibitors or phenotypic differences in
mature endothelium. It is not known whether the exogenous VEGF crossed
the placenta to reach the developing fetus, but no vascular defects
were observed in the embryos from VEGF-treated mice. However, direct
injection of exogenous VEGF into Quail embryos induced malformed and
hyperfused vessels during embryonic neovascularization (36). These data
suggest that developing organs are more sensitive to the change of
balance of angiogenic inducers and inhibitors.
The expression of FLT-1 and/or sFLT-1 by alternative splicing of FLT-1
pre-mRNA in the spongiotrophoblast cells provides a means by which they
can regulate the local response to VEGF. How the switch between FLT-1
and sFLT-1 is regulated needs to be further elucidated. Understanding
this will provide a fuller understanding of physiological angiogenesis
and may lead to novel means for the modulation of pathological
angiogenesis.
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MATERIALS AND METHODS
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Tissue and Serum Collection
All procedures and care of animals were in accordance with the
regulations laid down by the UK Home Office. BALB/c females (2025 g)
were mated with males of the same stock, and the day the vaginal plug
was detected was designated day 1 of pregnancy. Once pregnant, the mice
were housed individually. Placentas were collected on days 11, 13, 15,
and 17 of pregnancy. Organs from adult mice (1012 weeks old),
including lung, liver, kidney, and uterus, were also collected. Tissues
were fixed overnight in 10% formalin in PBS and processed for routine
histology or snap-frozen in liquid nitrogen and stored at -70 C.
Twenty placentas from day 17 pregnant mice were minced, washed three
times in DMEM/nut mix F-12 medium (GIBCO/BRL), and cultured in the same
medium at 37 C in 5% CO2. Conditioned medium was harvested
24 h later and stored at -20 C for further analysis.
VEGF treatment commenced on day 9 of gestation and continued until day
17. Recombinant human VEGF (Amgen, endotoxin <0.006 pg/µg VEGF) was
dissolved in PBS and 1.5 µg were injected ip into the pregnant mice
(n = 5) daily. Control mice (n = 4) were treated with vehicle
alone. At autopsy on day 18, the number of resorption sites per mouse
was determined and the embryo and placenta were weighed. Tissue was
collected and processed as above and 5-µm sections were cut and
stained for fibrin using the specific histological stain Martius
Scarlet Blue (MSB) (37) in which fibrin is stained bright red.
Blood from normal pregnant and nonpregnant mice was also collected.
Serum was recovered and stored at -70 C until required for
analysis.
3'-RACE
Total RNA was isolated from placenta of day 17 pregnant mice
using the method of Chomczynski and Sacchi (38). Poly (A)+ RNA was purified using oligo (dT) cellulose (Pharmacia,
Piscataway, NJ) and cDNA was prepared using (dT) 17-adaptor primer and
Super RT (HT Biotechnology Ltd, Cambridge, UK) following the
manufactures instructions. The (dT)17-adaptor primer
(GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT) was according to Frohman et
al. (39). A portion of this cDNA was used to amplify the desired
3'-end sequence of mouse sFLT-1 using a gene-specific primer (GSP:
GCCAGGAACATATACACAG) corresponding to bases 19411959 of mouse FLT-1
(40), and an adaptor primer (GACTCGAGTCGACATCGA), with a HYBAID
Touchdown thermocycler in 20 µl of PCR cocktail comprising
deoxynucleoside triphosphates (200 µM), 1x BioTaq
polymerase buffer, Mg++ solution (1.5 mM), 1 U
of BioTaq DNA polymerase (Bioline, London, UK). The reaction
mixture was denatured at 95 C for 2 min, and then amplified for 35
cycles (95 C, 30 sec; 52 C, 1 min; 72 C, 40 sec), followed by a 3-min
final extension at 72 C. After end repair, the PCR products were cloned
in pCR-Script Amp SK (+) cloning vector (Stratagene, La Jolla, CA) and
sequenced.
Probes
To generate probes for the specific detection of sFLT-1 or FLT-1
transcripts, or both, RT-PCR was performed to produce cDNA fragments
using specific primers. A 105-bp long 3'-end cDNA fragment of mouse
sFLT-1, which starts from the divergent site of FLT-1 and sFLT-1, was
generated by RT-PCR using mRNA extracted from placenta of day 17
pregnant mice. The primers used were based on the unique 3'-end
nucleotide sequence of mouse sFLT-1 obtained as described above. The
5'-primer was AGG TGA GCA CTG CGG CA (msflt-1), and the 3'-primer was
ATG AGT CCT TTA ATG TTT GAC (msflt-2). A cDNA fragment corresponding to
bases 37994011 of mouse FLT-1 nucleotide sequence (40), which showed
little similarity to the other members of the receptor tyrosine kinase
family, was also generated by RT-PCR as described above. The 5'-primer
was (3799) TCA CCT GGA CTG AGA CCA AG (3819) and the 3'-primer was
(3990) GTA CAA CAC CAC GGA GTT GTA (4011). Another cDNA fragment
corresponding to bases 11210 of mouse FLT-1 nucleotide sequence (40),
which detects both sFLT-1 and FLT-1 transcripts, was also generated as
described above. The 5'-primer was 11 CCG CGT CTT GCT CAC CAT G 29, and
the 3'-primer was 210 ACC ATG AGT GGG CTG CCT C 192. cDNA fragments
generated above were cloned in pCR-Script Amp SK(+) vector and
sequenced.
In Situ Hybridization
In situ hybridization was carried out essentially as
described by Clark et al. (31). Hybridization probes used
were specific for detecting sFLT-1 or membrane-bound FLT-1 transcripts.
This was confirmed by Northern blot analysis of total RNA from day 15
placenta (Fig. 2
, A and B). To generate RNA probes for in
situ hybridization, the constructs described above were linearized
by digestion with the appropriate restriction endonuclease, and
33P-UTP (Amersham International PLC, Little Chalfont,
U.K.)-labeled sense and antisense RNA probes were synthesized by
in vitro transcription using T7- and T3-polymerases
respectively (Ambion, Inc., Austin, TX).
Northern Blot Analysis
Total RNA (20 µg) was separated in a 1% (wt:vol) agarose/6%
formaldehyde (wt:vol) gel prepared in 1x MEA buffer (20 mM
3-(N-morpholino)propane sulfonic acid, 5
mM NaAc, 1 mM EDTA, pH 7.O), transferred to
nylon membrane (Amersham) by capillary blot, and fixed by UV
cross-linking. The blot was then prehybridized in 50 ml of
hybridization buffer comprising 50 mM Tris-HCl (pH 7.6),
0.1% (wt/vol) SDS, 10x Denhardts [0.2% (wt/vol) Ficoll, 0.2%
(wt/vol) PVP, 0.2% (wt/vol) BSA], 0.1% sodium pyrophosphate
(wt/vol), 6% polyethylene glycol (PEG 6000) (wt/vol), 6% NaCl
(wt/vol), and 0.1 mg/ml salmon sperm DNA at 65 C for 2 h. Probes
specific for sFLT-1 or FLT-1, or both transcripts, were prepared using
cDNA fragments generated above and T7 QuickPrime Kit (Pharmacia
Biotech) following the manufacturers instructions. Probe was
labeled to a specific activity of 12 x 106 cpm/ng
using [a32P] dCTP (Amersham). Before addition to the
hybridization solution, the probe was denatured by boiling for 3 min.
The incubation was continued at 65 C overnight. The blot was then
washed in 1x SSC/0.1% (wt/vol) SDS at 65 C with three changes of
washing buffer and wrapped in Saran wrap for autoradiography. The
exposure was performed at -70 C overnight using double-coated x-ray
film (Fuji Medical Systems, Stamford, CT) in conjunction with
intensifying screens. The film was developed in an x-ograph automatic
film developer.
RT-PCR
Extraction of total RNA from lung, liver, kidney, and uterus and
preparation of cDNA were performed as already described. Similar
amounts of RNA from each sample were used for RT and amplification. The
specific sFLT-1 primers were msflt-1 and msflt-2 described above. The
PCR program was as follows: 95 C for 1 min; 28 cycles (95 C, 30 sec; 54
C, 30 sec; 72 C, 30 sec); followed by a 3-min final extension at 72 C.
RNA without RT was used as negative control.
Western Blot Analysis
Human sFLT-1 has been shown to bind heparin Sepharose (26). We
therefore used this affinity matrix to partially purify the
VEGF-binding activity in mouse placenta-conditioned medium. Conditioned
medium from 20 mouse placentas (15 ml) was centrifuged and then loaded
onto a HiTrap heparin 1-ml column (Pharmacia) equilibrated in PBS. The
column was washed with 5 ml of PBS and 5 ml of 0.6 M
NaCl/20 mM phosphate buffer (pH 7.4), and the activity was
eluted with 1.2 M NaCl in the same buffer. The elute was
then concentrated using Centricon-30 (Amicon, Inc., Beverly, MA) and
washed in PBS. A portion of this was electrophoretically separated by
412% Bis-Tris NuPAGE (Novex, San Diego, CA) and transferred to
nitrocellulose membrane (Amersham). The membrane was blocked with 5%
BSA in PBS/0.1% Tween 20/0.1 M NaCl and probed with
biotinylated anti-human FLT-1 antibody (0.15 µg/ml) (R&D Systems,
Minneapolis, MN) in the same buffer containing 1% BSA. Immunoreactive
bands were detected using streptavidin-horseradish peroxidase
(Amersham) and Supersignal substrate (Pierce, Rockford, IL).
S-200 Gel Filtration Chromatography
Serum (50 µl) from nonpregnant and pregnant mice on days 11,
13, 15, and 17 of gestation (n = 3 for each stage) was incubated
with 0.7 ng of 125I-labeled human VEGF (2300 Ci/mmol,
Amersham), respectively, at room temperature overnight. Day 17 pregnant
mouse serum in the presence of 100-fold excess of unlabeled recombinant
human VEGF, or nonpregnant mouse serum in the presence of
baculovirus-expressed recombinant human sFLT-1 (1 ng), was also
incubated with the same amount of [125I]VEGF. Samples
were analyzed by loading onto a 12.5-ml Sephacryl S-200 (Sigma Chemical
Co., St. Louis, MO) gel column preequilibrated in PBS as described by
Hill et al. (41). Forty fractions eluted in PBS, each of 460
µl, were collected, and the radioactivity of each fraction was
counted using a
-counter (Packard Instruments, Meriden, CT).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Andrew Sharkey for helpful discussion of this work
and Ms. Amanda Evans for her assistance with DNA sequencing.
 |
FOOTNOTES
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Address requests for reprints to: Mr. Yulong He or Dr. D. Stephen Charnock-Jones, Department of Obstetrics and Gynaecology, University of Cambridge, The Rosie Hospital Reproductive Molecular Research Group, Robinson Way, Box 223, Cambridge, UK CB2 2SW .E-Mail: yh1{at}mole.bio.cam.ac.uk or dscj1{at}cam.ac.uk
D.S.C.J. was supported in part by Biotechnology and Biological Sciences
Research Council (Fellowship PDF/22).
Received for publication June 1, 1998.
Revision received December 29, 1998.
Accepted for publication January 4, 1999.
 |
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