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GOOD LUCK.
A HUMAN-SPECIFIC SOLUBLE VEGF-RECEPTOR 1:
ABSTRACT
A human-specific splicing variant of VEGF receptor 1 (Flt1) was discovered, producing a
soluble receptor (designated” sFlt1-14”) that is qualitatively different from the previously
described soluble receptor (sFlt1), and functioning as a potent VEGF inhibitor. sFlt1-14 is
generated in a cell type-specific fashion, primarily in non-endothelial cells.. Increased production
of soluble VEGF receptors during pregnancy is entirely due to induced expression of placental
sFlt1-14 starting by the end of the first trimester. Expression is dramatically elevated in the
placenta of women with preeclampsia (PE), specifically induced in abnormal clusters of
degenerative syncytiotrophoblasts known as syncytial knots where it may undergo further
mRNA editing. sFlt1-14 is the predominant VEGF-inhibiting protein accumulating in the
circulation of preeclampsic women and hence capable of neutralizing VEGF in distant organs
affected in PE. Together, these findings revealed a new natural VEGF inhibitor that has evolved
in humans, possibly to protect non-endothelial cells from adverse VEGF signaling. Further, the
study uncovered the exact identity of the VEGF-blocking protein implicated in PE.
2
INTRODUCTION
VEGF is the key factor promoting vasculogenesis and angiogenesis in the embryo and the factor
orchestrating most, if not all processes of adult neovascularization. In addition, VEGF performs
non-vascular developmental functions and plays a number of homeostatic roles in the adult. The
later includes maintenance of endothelial fenestrations, a role in vasodilatation, neurogenic and
neurotrophic activities, and others 1,2. Not surprisingly, therefore, VEGF is under a tight spatial
and temporal regulation and even a moderate deviation from its exquisite dosage control or
perturbation of its precise morphogenetic gradients are detrimental for proper development and
organ homeostasis3-6.
Among the multiple layers of VEGF control, natural VEGF inhibitors, primarily soluble VEGF
receptors, are likely to be important. Indeed, VEGF receptor-1 (Flt1) mRNA may undergo
alternative splicing in a way that the encoded protein retains the ligand-binding domain but is
devoid of the membrane spanning- and intracellular kinase domains, hence functioning as a
VEGF-trapping soluble receptor. This secreted protein (designated sFlt1) is a potent inhibitor of
both VEGF-A, VEGF-B and PLGF 2 . It is widely used as a research tool for VEGF inhibition
and more recently also harnessed in clinical trials of VEGF blockade. Surprisingly, however,
little is known concerning natural sFlt1 regulation. Likewise, the different areas where it may act
in physiologic titration of VEGF or in pathologic neutralization of VEGF activity remain to be
uncovered. A remarkable recent example for a physiologic role of sFlt1 is the identification of
sFlt1 as the factor responsible for preventing vascular invasion onto the cornea 7. The best
documented case of a pathological role is the involvement of sFlt1 in preeclampsia 8,9.
3
Preeclampsia (PE), affecting 5% of pregnant women is characterized by severe maternal
hypertension, proteinuria, glomerular endotheliosis and neurological symptoms. While the
underlying pathogenetic mechanism of PE is largely unknown, sFlt1 was implicated in PE
causation because of its abnormally high levels in the circulation of preeclempsic women 9.
Further, a seminal study by Maynard et. al. has shown that systemic sFlt1 causes a PE-like
syndrome in rats, presumably through negating constitutive VEGF function in the kidney 8.
Initially considered as an endothelial cell-specific receptor, Flt1 was subsequently detected also
in a number of non-endothelial cells, thus extending the spectrum of cells directly responsive to
VEGF. We reason that the splicing ratio of Flt1/sFlt1 should determine the extent to which a
given cell will transmit or, conversely, resist VEGF signaling. We propose that dynamic changes
in the splicing ratio may serve a regulatory function, in general, and that predominance of the
soluble receptor might protect non-endothelial cells from adverse effects of VEGF, in particular.
Experiments reported here are compatible with the proposition of a role for sFlt1 in the
EC/VSMC inter-phase of the mature vessel wall. We describe a novel splice variant of Flt1
functioning as a potent VEGF-blocking soluble receptor. This natural VEGF inhibitor designated
sFlt1-14, evolved during primate evolution and is the predominant VEGF inhibitor produced by
human non-endothelial cells. Importantly, we show that sFlt1-14 is also the VEGF-neutralizing
protein produced by the placenta of women with preeclampsia.
4
MATERIALS & METHODS
cDNA cloning. Placental RNA was prepared and converted to cDNA using standard procedures.
Rapid Amplification of cDNA Ends (RACE) was performed using a BD Smart RACE cDNA
Amplification Kit (Cat. no. 634914). For cloning/sequencing of sFlt1-14 3’-UTRs, RNAs of PE
placentas were reverse-transcribed using a primer complementary to the beginning of exon 14 of
Flt1.
Recombinant sFlt1 and sflt1-14 proteins. cDNAs encompassing the entire coding region of
both soluble receptor isoforms were sub-cloned into Bluescript expression vectors and
transfected onto T7 polymerase-expressing Hela cells. 20-24 hours later, media were collected
and the cells were harvested. Secreted and cell associated proteins were immuno precipitated
with the FLT11 (sigma) antibody and analyzed by immuno blotting with the ab9540 antibody
(abcam), both antibodies directed against the extra cellular domain of Flt1.
VEGF inhibition assay- Porcine Aortic Endothelial (PAE) cells engineered to express high
levels of human VEGF-R2 were exposed to VEGF pre-incubated (or not) with sFlt1 or sFlt1-14
proteins. A reduction in VEGF-R1 phosphorylation was determined using antibodies detecting
phospho-VEGF-R2 (Cell-signaling, Cat. #2478) and standardized to total VEGF-R2 protein
visualized by immunoblotting with anti-VEGF-R2 antibody (Santa Cruz Cat. SC-504).
RNA and protein analyses. For Northern-blot analysis, total RNA (5−20 μg) was resolved by
formaldehyde−agarose (1%) denaturing gels, blotted onto positively charged nylon membrane by
capillary elution and hybridized with
32
P-labeled specific cDNA probes prepared with the aid of
a Rediprime kit (Amersham). Probes derived from different regions of FLT1 transcripts were
used, as specified in the text and figure legends. For in situ detection of sFlt1-14 RNA, paraffin-
5
embedded sections of normal and PE placenta were hybridized with a 35S-labeled riboprobe
composed of intron 14 sequences as previously described (Motro et al., PNAS, 1990,
87(8),3092-6).
For IP/IB analysis, placentas were homogenized and immuno-precipitated with one of three
antibodies: CHFK produced in our laboratory , CESS produced in our laboratory and FLT11
(Sigma Cat. #V4262) and incubated overnight with Protein A-coated beads (P3391, Sigma).
Precipitates were than dissolved, proteins separated on 6% acrylamide gel, electrophoreticaly
transferred to a membrane, and detected with the CESS antibody. For analysis of serum
proteins, 20 ml serum from PE patients was first concentrated through capture on FLT11-coated
beads and affinity-purified proteins analysed by Western-blotting as described above. Placental
samples were taken with IRB approval. Preeclampsia samples met ACOG criteria for severe
preeclampsia.
Mass spectrometry – Protein homogenate of a PE placenta was incubated for 3 hours with a
rabbit pre-immune serum (20 l), incubated overnight with Protein A beads and precipitated by
through 3 hrs incubation with 15l of the CESS antibody. Preciptated proteins were
subsequently separated on a 6% acrylamide gel and visualized by staining with h coomassie
blue. A 115 Kd band was cut-out, digested with trypsin and was subjected to mass spectrometry
analysis by LC-MS/MS on DECA/LCQ. Peptides were identified and analyzed by Pep-Miner
and Sequest software against nr database of Human, mouse, rat bovine and Rabbit.
ELISA- A commercial ELISA kit for the detection of all soluble VEGF receptors isoforms was
used (R&D systems, DVR 100B).
Cells and culture- Human primary endothelial and vascular smooth muscle cells were a gift
from Dr. ,,,,
6
RESULTS
Identification of a novel splice variant of a soluble VEGF receptor-1 (sFlt1-14).
The majority of mRNA molecules transcribed from VEGF-R1 DNA encodes a transmembrane
signaling receptor but a small fraction of primary transcripts is usually alternatively spliced
generating a truncated mRNA devoid of the transmembrane- and intracellular-encoding domains
(Fig.1) and hence capable of sequestering VEGF and block signaling.. 3’-RACE methodology
was thus used for cloning truncated Flt1 mRNAs present in human placental RNA. These studies
culminated in the isolation and characterization of a novel human-specific splice variant of
VEGF-R1, designated therein as sFlt1-14 (Fig.1A).. sFLT1-14 sequence new data have been
submitted to the GenBank databases under accession No. EU368830.
We have found a new soluble sFlt1(which we called sFlt14)variant different in sequence from
the sFlt-1of the prior art (Maynard et al) and produced by the Flt gene by a previously undescribed mode of alternative splicing . This variant excludes intron 13 (present in the prior art
sFlt-1 but not in the full membranal receptor); includes exon 14 (present in the full membranal
Flt1 protein but not in the prior art soluble sFlt-1(-marked in the figure below as
UNDERLINED) and in addition includes sequences derived from intron 14 which are not
present either in the prior art sFlt -1 nor in the full membranal receptor(sequences from
expressed intron 14 are marked in the sequence below as BOLD )
Met V S Y W D T G V L L C A L L S C L L L T G S S S G S K L K D P E L S L K G T Q H I
MQAGQTLHLQCRGEAAHKWSLPEMVSKESERLSITKSACGRN
GKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFI
SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFP
7
LDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYK
TNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTR
VQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQN
KDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLET
VAGKRSYRLSMKVKAFPSPEVVWLKDGLPATEKSARYLTRGY
SLIIKDVTEEDAGNYTILLSIKQSNVFKNLTATLIVNVKPQIYEK
AVSSFPDPALYPLGSRQILTCTAYGIPQPTIKWFWHPCNHNHSE
ARCDFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKMAST
LVVADSRISGIYICIASNKVGTVGRNISFYITDVPNGFHVNLEK
MPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRTMHYSISKQK
MAITKEHSITLNLTIMNVSLQDSGTYACRARNVYTGEEILQKK
EITIRDQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWF
K N N H K I Q Q E P E L Y T S T S P S S S S S S P L S S S S S S S S S S S S Stop
The following is the cDNA sequence coding for the full sFlt14 is: (Bold and underlinedsequence coding for the expressed intron 14 )
ATGGTCAGCTACTGGGACACCGGGGTCCTGCTGTGCGCGCTGCTCAGCTGTC
TGCTTCTCACAGGATCTAGTTCAGGTTCAAAATTAAAAGATCCTGAACTGAGTTT
AAAAGGCACCCAGCACATCATGCAAGCAGGCCAGACACTGCATCTCCAATGCA
8
GGGGGGAAGCAGCCCATAAATGGTCTTTGCCTGAAATGGTGAGTAAGGAAAGC
GAAAGGCTGAGCATAACTAAATCTGCCTGTGGAAGAAATGGCAAACAATTCTGC
AGTACTTTAACCTTGAACACAGCTCAAGCAAACCACACTGGCTTCTACAGCTGC
AAATATCTAGCTGTACCTACTTCAAAGAAGAAGGAAACAGAATCTGCAATCTATA
TATTTATTAGTGATACAGGTAGACCTTTCGTAGAGATGTACAGTGAAATCCCCG
AAATTATACACATGACTGAAGGAAGGGAGCTCGTCATTCCCTGCCGGGTTACG
TCACCTAACATCACTGTTACTTTAAAAAAGTTTCCACTTGACACTTTGATCCCTG
ATGGAAAACGCATAATCTGGGACAGTAGAAAGGGCTTCATCATATCAAATGCA
ACGTACAAAGAAATAGGGCTTCTGACCTGTGAAGCAACAGTCAATGGGCATTTG
TATAAGACAAACTATCTCACACATCGACAAACCAATACAATCATAGATGTCCAAA
TAAGCACACCACGCCCAGTCAAATTACTTAGAGGCCATACTCTTGTCCTCAATT
GTACTGCTACCACTCCCTTGAACACGAGAGTTCAAATGACCTGGAGTTACCCTG
ATGAAAAAAATAAGAGAGCTTCCGTAAGGCGACGAATTGACCAAAGCAATTCCC
ATGCCAACATATTCTACAGTGTTCTTACTATTGACAAAATGCAGAACAAAGACAA
AGGACTTTATACTTGTCGTGTAAGGAGTGGACCATCATTCAAATCTGTTAACAC
CTCAGTGCATATATATGATAAAGCATTCATCACTGTGAAACATCGAAAACAGCA
GGTGCTTGAAACCGTAGCTGGCAAGCGGTCTTACCGGCTCTCTATGAAAGTGA
AGGCATTTCCCTCGCCGGAAGTTGTATGGTTAAAAGATGGGTTACCTGCGACT
GAGAAATCTGCTCGCTATTTGACTCGTGGCTACTCGTTAATTATCAAGGACGTA
ACTGAAGAGGATGCAGGGAATTATACAATCTTGCTGAGCATAAAACAGTCAAAT
GTGTTTAAAAACCTCACTGCCACTCTAATTGTCAATGTGAAACCCCAGATTT
ACGAAAAGGCCGTGTCATCGTTTCCAGACCCGGCTCTCTACCCACTGGGCAGC
AGACAAATCCTGACTTGTACCGCATATGGTATCCCTCAACCTACAATCAAGTGG
TTCTGGCACCCCTGTAACCATAATCATTCCGAAGCAAGGTGTGACTTTTGTTCC
AATAATGAAGAGTCCTTTATCCTGGATGCTGACAGCAACATGGGAAACAGAATT
GAGAGCATCACTCAGCGCATGGCAATAATAGAAGGAAAGAATAAGATGGCTAG
CACCTTGGTTGTGGCTGACTCTAGAATTTCTGGAATCTACATTTGCATAGCTTCC
AATAAAGTTGGGACTGTGGGAAGAAACATAAGCTTTTATATCACAGATGTGCCA
AATGGGTTTCATGTTAACTTGGAAAAAATGCCGACGGAAGGAGAGGACCTGAA
ACTGTCTTGCACAGTTAACAAGTTCTTATACAGAGACGTTACTTGGATTTTACTG
CGGACAGTTAATAACAGAACAATGCACTACAGTATTAGCAAGCAAAAAATGGCC
9
ATCACTAAGGAGCACTCCATCACTCTTAATCTTACCATCATGAATGTTTCCCTGC
AAGATTCAGGCACCTATGCCTGCAGAGCCAGGAATGTATACACAGGGGAAGAA
ATCCTCCAGAAGAAAGAAATTACAATCAGAGATCAGGAAGCACCATACCT
CCTGCGAAACCTCAGTGATCACACAGTGGCCATCAGCAGTTCCA
CCACTTTAGACTGTCATGCTAATGGTGTCCCCGAGCCTCAGATC
ACTTGGTTTAAAAACAACCACAAAATACAACAAGAGCCTGAACT
GTATACATCAACGTCACCATCGTCATCGTCATCATCACCATTGTC
ATCATCATCATCATCGTCATCATCATCATCATCATAG
.Notably, sFlt1-14 contains unique coding, as well as non-coding sequences shared neither by the
full-length receptor nor by the known soluble receptor, sFlt1. This was utilized for preparing
sFlt1-14-specific RNA probes that could be used for in situ hybridization having the following
sequence:
AACTGTATACATCAACGTCACCATCGTCATCGTCATCATCACCATTGTCATCATCATC
ATCATCGTCATCATCATCATCATCATAGCTATCATCATTATCATCATCATCATCATCA
TCATCATAGCTACCATTTATTGAAAACTATTATGTGTCAACTTCAAAGAACTTATCCT
TTAGTTGGAGAGCCAAGACAATCATAACAATAACAAA
and for preparation of sFlt1-14-specific antibodies directed against predicted carboxy-terminal
unique epitopes
One antibody was directed against the following sequence :
QEAPYL L R N L S D H T V A I S S S T T L D C H A N G V P E P Q I T W F K N N H K
IQQEP
which is the sequence of exon 14 (present in the full membranal Flt1 protein but not in the
prior art soluble sFlt-1
A second antibody was prepared against sequence
10
ELYTSTSPSSSSSSPL
Which is part of the sequence expressed intron 14 which are not present either in the prior art
sFlt -1 nor in the full membranal receptor
These antibodies were subsequently used to demonstrate that sFlt1-14 mRNA is naturally
translated into a 732 amino acid-long soluble receptor protein (see Fig.6 below).
sFlt1-14 is a natural VEGF inhibitor.
Considering that sFlt1 and sFlt1-14 are qualitatively different proteins (with sFlt1-14
containing 75 amino acids not present in sFlt1 and with sFlt1 containing 31 highly-conserved
amino acids not present in sFlt1-14), it was essential to demonstrate that sFlt1-14 functions as a
VEGF inhibitor. To this end, we first expressed a recombinant sFlt1-14 protein in human Hela
cells (and for comparison, also produced sFlt1-expressing Hela cells). Amounts of the respective
protein released to growth medium were then monitored by ELISA directed against a shared
extra-cellular epitope. For both proteins, concentrations of 100-200ng/ml were detected in the
medium. Media were further analyzed by immuno-precipitation and western blotting with two
different shared (extra-cellular) antibodies, confirming the mutually exclusive production of
either sFlt1-14 or sFlt1. Two sFlt1-14 proteins were detected - a cell-associated 115Kd isoform
and a secreted 130Kd isoform, likely representing additional post-translational modifications. As
expected from the size of the respective coding region, sFlt1-14 proteins were larger than the
corresponding secreted- and cellular sFlt1 isoforms (Fig.2A). To determine whether sFlt1-14
inhibits VEGF signaling, increasing amounts were pre-incubated with a constant amount of
VEGF (20ng/ml) prior to adding to the growth medium of Porcine Aortic Endothelial (PAE)
cells engineered to express high levels of human VEGF-R2. Levels of VEGF-R2
11
phosphorylation were than measured as a function of the sFlt1-14/VEGF ratio. As shown in
Fig.2B, sFlt1-14 inhibited VEGF-R2 phosphorylation almost completely already at a 1:1 ratio.
For comparison, at this ratio, sFlt1 did not significantly inhibit VEGF-R2 phosphorylation,
which was only evident at higher sFlt1/VEGF ratios. We conclude that sFlt1-14 is a potent
inhibitor of VEGF signaling, at least as good, if not better than sFlt1.
sFlt1-14 is upregulatd in syncytial knots of the preclampsic placenta
Previous studies highlighted the role of sFlt1 in preeclampsia (PE) causation, based on findings
that sFlt1 accumulates the circulation of women with PE to substantially higher levels than in
normal pregnancies and on findings that when expressed in rats, a chimeric protein containing
ligand-binding domains of sFlt1 produces the PE-like symptoms of hypertension and proteinuria
8
. Yet, the placental cellular sources of circulating VEGF receptors and factors responsible for its
deregulated expression in the human preeclampsia are not known. Furthermore, with the
discovery of sFlt1-14, the question arises which soluble receptor is associated with PE. To
address these questions, Placental RNAs from different stages of normal pregnancies and from
term placentas of both normal and PE pregnancies were first examined by northern blot analysis
with shared- and specific riboprobes. While expression of total soluble VEGF-R1 increases
progressively, particularly towards the third trimester of pregnancy, the relative contribution of
the two sFlt1 isoforms changed dramatically, with sFlt1-14 becoming the dominant isoform over
time. Thus, while the ‘classical’ sFlt1 accounted for approximately half of placenta-produced
soluble receptors during the first trimester, further increase at later times was entirely due to
upregulated expression of sFlt1-14 (Fig.4A). Transition to almost exclusive production of sFlt1-
12
14 took place by the beginning of the second trimester and was clearly evident at term (Fig.4A).
sFlt1-14 mRNA was also the dominant, if not the sole transcript encoding soluble Flt1 in the PE
placenta, reaching steady state levels exceedingly higher than in the term normal placenta
(Fig.4B). Identity of the respective mRNAs was secured through re-probing with a sFlt1-14specific riboprobe (data not shown) and was further confirmed by direct sequencing of cDNA
clones prepared from placenta RNA of 7 different PE patients.
To identify which cells in the PE placenta produce sFlt1-14, we carried-out in situ hybridization
analysis using a sFlt1-14-specific riboprobe
AACTGTATACATCAACGTCACCATCGTCATCGTCATCATCACCATTGTCATCATCATC
ATCATCGTCATCATCATCATCATCATAGCTATCATCATTATCATCATCATCATCATCA
TCATCATAGCTACCATTTATTGAAAACTATTATGTGTCAACTTCAAAGAACTTATCCT
TTAGTTGGAGAGCCAAGACAATCATAACAATAACAAA
(Fig.5). The major sites of placental sFlt1-14 expression were found to be syncytial knots, i.e.,
clusters of degenerative syncytiotropoblasts that are a histological hallmark of the PE placenta.
Noteworthy, sFlt1-14 is also produced by seemingly healthy syncytiotrophoblasts but at
significantly lower levels and, importantly, not at all in vascular endothelial cells (Fig.5 Right
and at a higher magnification in the image on the right), thus identifying syncytial knots as the
predominant site of sFlt1-14 production. It should be pointed-out that syncytial knots are often
found in aged normal placenta and we could indeed detect sFlt1-14 expression in syncytial knots
of normal placenta (data not shown). We conclude that the much increased abundance of
syncytial knot in PE accounts for the greatly elevated levels of sFlt1-14 produced in the PE
placenta.
13
sFlt1-14 is the predominant circulating VEGF-inhibiting protein in PE.
Findings that the PE placenta expresses exclusively sFlt1-14 argue that this is the major soluble
receptor isoform capable of accumulating in the serum of PE patients. To show that sFlt1-14
protein is produced in the PE placenta and subsequently released into the circulation of PE
patients, the following experiments were performed. First, term placentas from normal and PE
pregnancies were examined for the relative abundance of soluble receptors, using immunoprecipitations (IP) and immuno-blotting (IB) with isoform-specific antibodies. As expected from
the mRNA expression data, sFlt1-14 protein was abundantly produced in term placenta (Fig.6A).
Second, proteins from a preeclampsia placenta were immuno-precipitated with the sFLT1-14
specific antibody directed against the expressed intron 14 (E L Y T S T S P S S S S S S P L)
and the eluted 115 Kd band was unequivocally identified as sFlt1-14 using mass-spectrometry.
Particularly informative in this regard was a peptide spanning the exon13-exon 14 junction
which is not present in sFlt1 (Fig.6B).
Third, sera of PE women were analyzed for soluble Flt1
receptors. ELISA with antibody directed against a shared extra-cellular domain confirmed earlier
report of significantly elevated levels of circulating soluble receptor in PE (800-900 pg/ml vs.
8000-10000 pg/ml in control and PE third trimester sera, respectively). To identify the molecular
identity of circulating soluble receptors (whether it is the Sftl1 or the new Sflt14), PE serum
specimens were passed through FLT1 antibody-coated columns and bound proteins were
subsequently analyzed by western blotting (Fig.6C). Again, sFlt1-14 protein was readily detected
in the PE serum, visualized as two bands identical in size to those produced by the cells
transfected with sFlt1-14 expression plasmid and detected with the sFlt1-14-specific antibody .
14
Notably, we failed to detect appreciable levels of sFlt1 in these sera in the limited set of patients
analyzed, indicating that sflt1-14 is the major VEGF –inhibiting protein in the PE circulation.
15
DISCUSSION
The study uncovered a new player in VEGF regulation namely, a novel variant of soluble VEGF
receptor-1 (designated sFlt1-14) functioning as a potent VEGF inhibitor. Evolutionarily, sFlt1-14
represents a relatively recent addition to the multiple layers of VEGF control, as the unique
splicing pattern generating it, utilizing a previously unrecognized intron 14 splice acceptor (SA)
site, only takes place in humans and possibly also in non-human primate harboring Alu insertion
within the Flt1 locus. ..
The newly-discovered sflt1-14 is qualitatively different from sFlt1 by both missing 31 amino
acids, and by containing 28 unique amino acids. Although the 31 amino acids missing in sFlt114 are evolutionary highly conserved, arguing for their functional importance, inhibition of
VEGF signaling was not compromised. Conversely, it is possible that addition of extra amino
acids (e.g. a polyserine stretch) confer unique biological properties to sFlt1-14. . The most
significant pathology in which a soluble VEGF-R1 has been causally implicated is preecempsia.
This was based on detecting abnormally high levels of soluble receptor in circulation of
preecelmpsic women and on demonstrating that its systemic administration in rats produces a
PE-like syndrome 8,9. Here we provide evidence that the soluble receptor produced by the
placenta, accumulating in the circulation of PE women, and hence capable of affecting distant
organs is predominantly, if not exclusively, sFlt1-14 and not the previously recognized sFlt1.
The discrepancy with published work is explained by the fact that, unlike in our study, previous
studies have only used probes and antibodies directed against the extra-cellular domain of Flt1,
i.e. against a sequence or an epitope shared by all mRNA and proteins encoded by Flt1 gene. The
facts that PE is a human-specific disease and that sFlt1-14 is a human-specific protein raise the
16
speculation that a unique aspect in sflt1-14 regulation or a unique, yet unidentified biological
property of this protein are responsible for disease. Yet, the reason why rodents do not develop
PE could also be attributed to inherent differences in placentation in rodents and primates. A
sFlt1-14-expressing mouse (currently under construction) should aid addressing this question.
Here we identified syncytial knots as the major source of local and circulating sFlt1-14. The fact
that these structures increase in number in the normal aging placenta but are much more
abundant in the degenerating PE placenta, a phenomenon known as the "Tenney-Parker
change"18, may explain findings that serum levels of soluble receptors (now identified as mostly
sFlt1-14) increase in the second and third trimesters of normal pregnancies but to dramatically
higher levels in PE pregnancies. These findings place syncytial knotting, a phenomenon common
to several placental pathologies, as central to PE pathogenesis. We note in this regard that
hypoxia, often considered to be an ethiological cause of PE, is known to induce syncytial
knotting 19 and that syncytial knotting is also increased in placental malaria 20 known to be
associated with increasing levels of circulating VEGF-R1 receptors 21.
Finally, we note that our finding provide another example for a required caution in
extrapolating from mouse systems to human disease, as the later might also involve humanspecific mRNAs and proteins.
17
REFERENCES
.1
Cao L, Jiao X, Zuzga DS, Liu Y, Fong DM, Young D, During MJ. VEGF links
hippocampal activity with neurogenesis, learning and memory. Nat Genet. 2004;36:82735.
.2
Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptor signalling - in
control of vascular function. Nat Rev Mol Cell Biol. 2006;7:359-71.
.3
Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M,
Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D,
Risau W, Nagy A. Abnormal blood vessel development and lethality in embryos lacking
a single VEGF allele. Nature. 1996;380:435-9.
.4
Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O'Shea KS, Powell-Braxton L,
Hillan KJ, Moore MW. Heterozygous embryonic lethality induced by targeted
inactivation of the VEGF gene. Nature. 1996;380:439-42.
.5
Miquerol L, Langille BL, Nagy A. Embryonic development is disrupted by modest
increases in vascular endothelial growth factor gene expression. Development.
2000;127:3941-6.
.6
Carmeliet P, Ng YS, Nuyens D, Theilmeier G, Brusselmans K, Cornelissen I, Ehler E,
Kakkar VV, Stalmans I, Mattot V, Perriard JC, Dewerchin M, Flameng W, Nagy A,
Lupu F, Moons L, Collen D, D'Amore PA, Shima DT. Impaired myocardial angiogenesis
and ischemic cardiomyopathy in mice lacking the vascular endothelial growth factor
isoforms VEGF164 and VEGF188. Nat Med. 1999;5:495-502.
18
.7
Ambati BK, Nozaki M, Singh N, Takeda A, Jani PD, Suthar T, Albuquerque RJ, Richter
E, Sakurai E, Newcomb MT, Kleinman ME, Caldwell RB, Lin Q ,Ogura Y, Orecchia A,
Samuelson DA, Agnew DW, St Leger J, Green WR, Mahasreshti PJ, Curiel DT, Kwan
D, Marsh H, Ikeda S, Leiper LJ, Collinson JM, Bogdanovich S, Khurana TS, Shibuya M,
Baldwin ME, Ferrara N, Gerber HP, De Falco S, Witta J, Baffi JZ, Raisler BJ, Ambati J.
Corneal avascularity is due to soluble VEGF receptor-1. Nature. 2006;443:993-7.
.8
Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, Libermann TA, Morgan JP,
Sellke FW, Stillman IE, Epstein FH, Sukhatme VP, Karumanchi SA. Excess placental
soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction,
hypertension, and proteinuria in preeclampsia. J Clin Invest. 2003;111:649-58.
.9
Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, Schisterman EF,
Thadhani R, Sachs BP, Epstein FH, Sibai BM, Sukhatme VP, Karumanchi SA.
Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med.
2004;350:672-83.
.10
Sorek R, Lev-Maor G, Reznik M, Dagan T, Belinky F, Graur D, Ast G. Minimal
conditions for exonization of intronic sequences: 5' splice site formation in alu exons.
Mol Cell. 2004;14:221-31.
.11
Chandra A, Angle N. Vascular endothelial growth factor stimulates a novel calciumsignaling pathway in vascular smooth muscle cells. Surgery. 2005;138:780-7.
.12
Wang Z, Castresana MR, Newman WH. Reactive oxygen and NF-kappaB in VEGFinduced migration of human vascular smooth muscle cells. Biochem Biophys Res
Commun. 2001;285:669-74.
19
.13
Lee S, Chen TT, Barber CL, Jordan MC, Murdock J, Desai S, Ferrara N, Nagy A, Roos
KP, Iruela-Arispe ML. Autocrine VEGF signaling is required for vascular homeostasis.
Cell. 2007;130:691-703.
.14
Iacono M, Mignone F, Pesole G. uAUG and uORFs in human and rodent 5'untranslated
mRNAs. Gene. 2005;349:97-105.
.15
Hughes TA. Regulation of gene expression by alternative untranslated regions. Trends
Genet. 2006;22:119-22.
.16
Levanon K, Eisenberg E, Rechavi G, Levanon EY. Letter from the editor: Adenosine-toinosine RNA editing in Alu repeats in the human genome. EMBO Rep. 2005;6:8315.-
.17
Yang JH, Luo X, Nie Y, Su Y, Zhao Q, Kabir K, Zhang D, Rabinovici R. Widespread
inosine-containing mRNA in lymphocytes regulated by ADAR1 in response to
inflammation. Immunology. 2003;109:15-23.
.18
Tenney B, Parker F. The Placenta in Toxemia of Pregnancy. Am J Obstet Gynecol.
1940;39:1000-1005.
.19
Heazell AE, Moll SJ, Jones CJ, Baker PN, Crocker IP. Formation of syncytial knots is
increased by hyperoxia, hypoxia and reactive oxygen species. Placenta. 2007;28 Suppl
A:S33-40.
.20
Crocker IP, Tanner OM, Myers JE, Bulmer JN, Walraven G, Baker PN.
Syncytiotrophoblast degradation and the pathophysiology of the malaria-infected
placenta. Placenta. 2004;25:273-82.
.21
Muehlenbachs A, Mutabingwa TK, Edmonds S, Fried M, Duffy PE. Hypertension and
maternal-fetal conflict during placental malaria. PLoS Med. 2006;3:e446.
20
FIGURE LEGENDS
Figure 1. Structure of the novel VEGF-R1 soluble receptor (sFlt1-14).
A. A schematic representation of mRNAs encoding the full-size trans-membrane receptor (Flt1),
the known soluble receptor (sFlt1) and the new alternatively-spiced soluble receptor (sFlt1-14).
Translation start and stop codons are indicated. Boxes mark the three different RNA probes used
in this work : A probe encompassing exons 1-5 detecting all three receptor isoforms, a probe
derived from exon 13 specific for sFlt1 and an intron 14-based probe specific for sFlt1-14.
B. The predicted amino acid sequence of sFlt1-14 protein, starting at exon 13 (aa 623 in
NP_002010). First raw depicts exon 13 amino acids (shared by all three receptors), second raw
show exon 14 amino acids (shared by flt1 and sFlt1-14 but absent in sFlt1), third raw show
exonized amino acids derived from intron 14 (unique to sFlt1-14). Note the unusual polyserine
stretch at the carboxy-terminus. The underlined amino acids served for developing exons 14 and
exon 14a specific antibodies, designated CHFK and CESS, respectively.
Figure 2. sFlt1-14 is a potent inhibitor of VEGF signaling.
A. Recombinant sFlt1 and sFlt1-14 proteins were produced in HeLa cells transfected with the
respective expression vector. Soluble flt1 receptors found in the cell (c) or released to the
medium (m) were detected by Western blotting using antibodies directed against shared
epitopes. Note the presence of a 130Kd secreted sFlt1-14 protein and a 115Kd sflt1-14 protein in
the cellular fraction.
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B. Porcine aortic Endotelial cells (PAE) stably expressing Flk1 were exposed to 20ng/ml VEGF
and Flk1 phosphorylation was determined as readout for the extent of VEGF signaling.
Inhibitory effect of pre-incubation of VEGF with the indicated amounts of recombinant sFlt1 or
sFlt1-14 was measured, presented as the pFlk1/Flk1 ratio relative to the non-inhibited ratio.
Figure 3. Entire figure and related text in article were taken out
Figure 4. Prefferential expression of sFlt1-14 in the maturing normal and PE placenta.
A. (Left) Northern blot analysis of placental RNAs from the indicated weeks of gestation with a
probe detecting shared sequences of sFlt1-14 and sFlt1. (Right) The sFlt1-14/sFlt1 expression
ratio calculated on the basis of hybridization intensities. Note dominance of sFlt1-14 from the
second trimester and onwards.
B. Northern blot analysis with the shared extra-cellular part of Flt1 identifies sflt1-14 as the
predominant receptor isoform in the PE placenta
Figure 4 (c )was taken out of the text
Figure 5. Identification of sFlt1-14-producing cell in the placenta. In situ hybridization with
a sFlt1-14-specific riboprobe. Note that syncytial knots (SK) are the major source of placental
sFlt1-14, with a lower expression by apparently healthy syncytiotrophoblasts (STB) and no sflt114 expression by placental endothelial cells (EC).
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Figure 6. sFlt1-14 protein produced by the placenta accumulates in the circulation of
preecempsia patients.
A. Protein extracts from term human placenta were immuno-precipitated separately with the
three indicated antibodies detecting each a different epitope of sFlt1-14 and immuno-blotted with
the antibody unique to sFlt1-14. This procedure identified the 130Kd and 115Kd placental
proteins as sFlt1-14.
B. The 115 Kd protein band immuno-precipitated from a preeclamptic placental biopsy with the
CESS antibody and visualized by coomassie blue staining, eluted and analyzed by mass
spectrometry. Highlighted in red are three identified peptides that map either to the first 13 exons
or to the exon13/exon14 junction (Junction indicated by the green vertical line). The latter ruleout that the protein is sFlt1, while the CESS immuno-precipitation and size considerations ruleout that it is Flt1, thus identifying the 115kd protein unequivocally as sFlt1-14.
C. 20ml serum of a PE patient were passed through a column of beads coated with anti-Flt1
antibodies (against a shared extra-cellular epitope), eluted at a low pH and immuno-blotted with
the sFlt1-14-specific CESS antibody. For comparison, a CESS immuno-precipitate of the
placenta was run in parallel and immunoblotted . Note that the same two protein bands detected
in the placenta (and identified above as sFlt1-14 proteins) are also found in the PE serum.
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