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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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.
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. 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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). 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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) 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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, 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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), 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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). 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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
manufacture’s 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 1941–1959 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 3799–4011 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 11–210 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 manufacturer’s instructions. Probe was
labeled to a specific activity of 1–2 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
4–12% 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).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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L. Huminiecki, H.Y. Chan, S. Lui, R. Poulsom, G. Stamp, A.L. Harris, and R. Bicknell Vascular endothelial growth factor transgenic mice exhibit reduced male fertility and placental rejection Mol. Hum. Reprod., March 1, 2001; 7(3): 255 - 264. [Abstract] [Full Text] [PDF]
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 C. Robinson and S. Stringer The splice variants of vascular endothelial growth factor (VEGF) and their receptors J. Cell Sci., January 3, 2001; 114(5): 853 - 865. [Abstract] [PDF]
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