Molecular Human Reproduction, Vol.23, No.2 pp.

69–78, 2017
Advanced Access publication on December 28, 2016 doi:10.1093/molehr/gaw077

NEW RESEARCH HORIZON Review

Placental-specific sFLT-1: role in

pre-eclamptic pathophysiology and
its translational possibilities for
clinical prediction and diagnosis

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K.R. Palmer1,2,*, S. Tong2, and T.J. Kaitu’u-Lino2
1
Department of Obstetrics and Gynaecology, Monash University, Monash Medical Centre, 246 Clayton Rd, Clayton, 3168 Victoria, Australia
2
Translational Obstetric Group, University of Melbourne, Mercy Hospital for Women, 163 Studley Rd, Heidelberg, 3084 Victoria, Australia

*Correspondence address. Department of Obstetrics and Gynaecology, Monash University, Monash Medical Centre, 246 Clayton Rd,
Clayton, 3168 Victoria, Australia. E-mail: kirsten.palmer@monash.edu
Submitted on August 10, 2016; resubmitted on November 16, 2016; editorial decision on December 2, 2016; accepted on December 9,
2016

ABSTRACT: Pre-eclampsia is a common obstetric complication globally responsible for a significant burden of maternal and perinatal mor-
bidity and mortality. Central to its pathophysiology is the anti-angiogenic protein, soluble fms-like tyrosine kinase-1 (sFLT-1). sFLT-1 is
released from a range of tissues into the circulation, where it antagonizes the activity of vascular endothelial growth factor and placental
growth factor leading to endothelial dysfunction. It is this widespread endothelial dysfunction that produces the clinical features of pre-
eclampsia including hypertension and proteinuria. There are multiple splice variants of sFLT-1. One, known as sFLT-1 e15a, evolved quite
recently and is only present in humans and higher order primates. This sFLT-1 variant is also the main sFLT-1 secreted from the placenta.
Recent work has shown that sFLT-1 e15a is significantly elevated in the placenta and circulation of women with pre-eclampsia. It is also bio-
logically active, capable of causing endothelial dysfunction and the end-organ dysfunction seen in pre-eclampsia. Indeed, the over-expression
of sFLT-1 e15a in mice recapitulates the pre-eclamptic phenotype in pregnancy. Therefore, here we propose that sFLT-1 e15a may be the
sFLT-1 variant primarily responsible for pre-eclampsia, a uniquely human disease. Furthermore, this placental-specific sFLT-1 variant provides
promise for use as an accurate biomarker in the prediction or diagnosis of pre-eclampsia.

Key words: pre-eclampsia / anti-angiogenic factors / VEGF / placenta / hypoxia / sFLT-1 / splice variant

relate to underlying maternal endothelial and vascular disease

Introduction
Pre-eclampsia is a common complication of pregnancy, affecting 5–8%
of all pregnancies. It is a major contributor to global maternal and peri-
natal morbidity and mortality, leading to ~60 000 direct maternal
deaths and ~500 000 perinatal deaths annually (WHO, 2005). Pre-
eclampsia is a clinical diagnosis, whereby a mother develops high blood
pressure and evidence of other end-organ disease, such as renal, liver,
haematological, respiratory, cerebral or fetoplacental impairment (Mol
et al., 2016). Indeed, this heterogeneity in presentation is reflected in
the broad criteria used for its diagnosis (Tranquilli et al., 2014).
The underlying pathophysiology of pre-eclampsia is still incompletely
understood. Early-onset disease (seen at <34 weeks gestation) is
thought to relate to abnormally shallow placental invasion leading to
persisting placental hypoxia and reperfusion injuries that increase oxi-
dative stress within the placenta (Steegers et al., 2010; Myatt and
Roberts, 2015). In comparison, late-onset pre-eclampsia is thought to
(Steegers et al., 2010; Myatt and Roberts, 2015). In both situations,
the clinical features of disease still likely result from the effects of circu-
lating anti-angiogenic factors released from the placenta.
Since 1694, the release of such a factor(s) from the uterus or pla-
centa into the mother’s circulation to cause pre-eclampsia has been
postulated (Chesley, 1984). While many candidate toxins have been
proposed, none had been able to experimentally reproduce the com-
plete pre-eclamptic phenotype in vivo when administered to animals:
that was until the finding that over-expression of soluble fms-like tyro-
sine kinase-1 (sFLT-1) could produce a pre-eclamptic phenotype in
pregnant rats (Maynard et al., 2003). This breakthrough discovery
initiated a decade of subsequent research, which has firmly cemented
a major role for sFLT-1 in the pathogenesis of pre-eclampsia.
sFLT-1 comprises the extracellular domains of the vascular endo-
thelial growth factor receptor-1 (VEGFR-1), and is soluble, being pre-
sent in the circulation. It acts as an anti-angiogenic protein by

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70
antagonizing the actions of both vascular endothelial growth factor
(VEGF) and placental growth factor (PlGF). Multiple splice variants of
sFLT-1 have been described (Sela et al., 2008; Heydarian et al., 2009;
Thomas et al., 2009). These have significantly different tissue distribu-
tions (Jebbink et al., 2011), raising the potential for different physiological
and pathological roles. For example, in humans, the main sFLT-1 variant,
known as sFLT-1 i13, is widely expressed in most tissues, whereas
another variant known as sFLT-1 e15a appears to be almost exclusively
expressed by the placenta (Jebbink et al., 2011).
Ongoing progression in the understanding of the pathophysiology of
pre-eclampsia is critical to assisting in improving the clinical care for
affected women. Through better understanding, improved approaches
for disease prediction, diagnosis and therapies can be developed.
Indeed, the identification of a placental-specific sFLT-1 variant holds
promise for better prediction and diagnosis of pre-eclampsia.
sFLT-1 and pre-eclampsia
The placenta is central to the development of pre-eclampsia. Indeed,
conditions with an increased placental mass, such as multiple and
molar pregnancies, carry an elevated risk for pre-eclampsia. Both con-
ditions have been shown to significantly increase sFLT-1 expression
(Bdolah et al., 2008; Kanter et al., 2010; Koga et al., 2010). Indeed,
most women with pre-eclampsia have significantly increased levels of
sFLT-1 present in their circulation (Levine et al., 2004; Shibata et al.,
2005; Levine et al., 2006; Palmer et al., 2015; Souders et al., 2015)
with levels dramatically falling within the 48 hours following delivery
(Chaiworapongsa et al., 2004; McKeeman et al., 2004) supporting the
notion that the placenta is the major source of sFLT-1 in pre-
eclampsia. However, despite the strong evidence that elevated levels
of sFLT-1 are associated with pre-eclampsia, this is not uniformly seen
in all established cases of pre-eclampsia (Levine et al., 2004; Powers
et al., 2005; Palmer et al., 2016a). This potentially relates to the het-
erogeneity of mechanisms via which pre-eclampsia may occur.
Early-onset pre-eclampsia is predominately thought of as a placental
disease due to poor placental implantation (Steegers et al., 2010),
while late-onset pre-eclampsia likely relates to pre-existing maternal
endothelial dysfunction (Ness and Roberts, 1996; Raymond and
Peterson, 2011; Myatt and Roberts, 2015). A postulated explanation is
the existence of an endothelial threshold for angiogenic factors. When
levels of sFLT-1 surpass this threshold, the net reduction in angiogenic
factors (VEGF and PlGF) leads to endothelial dysfunction and the clin-
ical presentation of pre-eclampsia (Maynard et al., 2003; Sugimoto
et al., 2003; Ahmad and Ahmed, 2004). In women with pre-existing
endothelial dysfunction (such as hypertension, obesity or diabetes) this
threshold is lowered, with less sFLT-1 required to produce the disease
state (Levine et al., 2006; Suwaki et al., 2006). In these circumstances,
the normal gestational rise that occurs in sFLT-1 (Levine et al., 2004;
Maynard et al., 2013; Palmer et al., 2015) may be enough for pre-
eclampsia to develop, although no significant increase in circulating
sFLT-1 during pregnancy is evident (Palmer et al., 2016a).
Total VEGF levels are also increased in pre-eclamptic pregnancies
(Clark et al., 1998; McKeeman et al., 2004) due to underlying placental
hypoxia. However, the amount of free, bioavailable VEGF is reduced
(Livingston et al., 2000; Maynard et al., 2003; Levine et al., 2004) due
to the greater concomitant rise in VEGF binding proteins, such as
sFLT-1. This was highlighted by Maynard et al. (2003), who
Palmer et al.
demonstrated an inversely proportional association between the levels
of sFLT-1 in the serum of pre-eclamptic women and serum concentra-
tions of VEGF and PlGF. They also demonstrated the impact of an
altered angiogenic balance on endothelial cell function. Their work
showed that human umbilical vein endothelial cells (HUVECs) exposed
to pre-eclamptic serum failed to form tubular structures (Maynard
et al., 2003), suggesting the presence of an anti-angiogenic factor within
the patient serum. Adding exogenous VEGF and PlGF to the pre-
eclamptic serum reversed this effect, further supporting this premise.
This was confirmed, as serum from healthy pregnant women induced
endothelial tube formation, an effect that was inhibited by the addition
of exogenous sFLT-1. The final confirmation of the involvement of
sFLT-1 in pre-eclampsia came with the adenoviral over-expression of
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sFLT-1 in pregnant rats. This produced a pre-eclamptic-like phenotype
of hypertension, proteinuria and glomerular endotheliosis (Maynard
et al., 2003). Importantly, these findings confirm that sFLT-1 alone is
capable of producing endothelial dysfunction. This work has subse-
quently been independently validated with sFLT-1 over-expression
producing a pre-eclamptic phenotype in animal models (Gilbert et al.,
2007; Makris et al., 2007; Bergmann et al., 2010; Kumasawa et al.,
2011).
In humans, sFLT-1 is significantly up-regulated within both placenta
and blood of women with pre-eclampsia (Ahmad and Ahmed, 2004;
Chaiworapongsa et al., 2004; Levine et al., 2004; McKeeman et al.,
2004; Powers et al., 2005), as is the placental-specific variant (Palmer
et al., 2015; Souders et al., 2015). Importantly, when sFLT-1 is
removed by either immunoprecipitation or apheresis, the pro-
angiogenic capabilities of the serum are restored (Ahmad and Ahmed,
2004) and the clinical phenotype in pregnant women stabilized
(Thadhani et al., 2016). Furthermore, aspirin, which is administered
clinically to prevent pre-eclampsia, has been shown to reduce sFLT-1
levels, including those of the placental-specific variant (Li et al., 2015).
Interestingly, a rise in serum sFLT-1 levels appears to precede the
onset of clinical disease, with levels significantly higher than normal
5 weeks prior to the clinical diagnosis of pre-eclampsia (Levine et al.,
2004; Powers et al., 2005). This provides encouragement for the use
of sFLT-1 levels in serum to predict the disease.
sFLT-1 acts as a VEGF trap
sFLT-1 belongs to the VEGF/VEGFR family; a family which plays a vital
role in angiogenesis and vasculogenesis. sFLT-1 was first identified by
Kendall and Thomas (1993), who described a soluble version of the
fms-like tyrosine kinase-1 (FLT-1) receptor in the media of HUVECs.
This was a time of rapid scientific advancement in the understanding of
angiogenesis; with both VEGF and the full-length membrane-bound
FLT-1 receptor (VEGFR-1) only identified in the preceding 4 years
(Leung et al., 1989; Shibuya et al., 1990). sFLT-1 is transcribed from the
FLT-1 gene, which is located on chromosome 13q12. The translated
sFLT-1 protein is identical to the majority of the extracellular region of
FLT-1, containing the first six out of seven Ig-like domains. This is
important, as the ligand-binding domain (second and third Ig-like
domains) is conserved, enabling both FLT-1 and sFLT-1 to bind VEGF
and PlGF with high affinity (Kendall et al., 1994; Park et al., 1994;
Wiesmann et al., 1997; Christinger et al., 2004). Furthermore, the
presence of the fourth Ig-like domain conserves the ability for receptor
dimerization and heparin binding (Wiesmann et al., 1997; Park and
Placental-specific sFLT-1
Lee, 1999). The presence of sFLT-1 in vivo was subsequently con-
firmed in the placenta and serum of pregnant women with pre-
eclampsia (Clark et al., 1998), where its presence antagonized the
actions of VEGF.
VEGF plays a crucial role in angiogenesis and normal endothelial cell
function. Its bioavailability is strictly regulated and alterations in its cir-
culating levels have a negative impact on endothelial cell function. This
is highlighted by the drastic phenotypes exhibited through the effects
of both under- and over-expression of VEGF on endothelial cell func-
tion in the kidney. Mice lacking Vegf solely within the podocytes (a vital
component of the glomerular basement membrane) die within
18 hours of birth due to hydrops, renal failure and abnormal glomeruli
(Eremina, 2003). Podocytes with heterozygous Vegf expression are
able to develop an appropriate glomerular filtration barrier (Eremina,
2003); however, their function is impaired with mice developing
endotheliosis (similar to that seen in pre-eclampsia) by 2.5 weeks of
age. These data support the premise that a reduction in VEGF bioavail-
ability in the kidney is capable of producing proteinuria (Eremina et al.,
2008). If reduced VEGF levels are maintained, gradual progression to
nephrotic syndrome with significant glomerular scarring occurs and
eventual loss of podocytes by 9 weeks of age (Eremina, 2003).
Similarly, a 2-fold increase in podocyte VEGF expression also produces
a dramatic phenotype with mice developing collapsing glomerulone-
phropathy, leading to death by 5 days of age (Eremina, 2003).
The use of VEGF antagonists, such as bevacizumab, in the treatment
of cancer has also highlighted the importance of VEGF in maintaining
vascular homoeostasis (Maharaj and D’Amore, 2007). The principal
side effects seen are hypertension and proteinuria; however arterial
thrombosis, cardiac ischaemia, gastrointestinal perforation, impaired
wound healing and reversible posterior leukoencephalopathy syn-
drome have all been reported (Glusker et al., 2006; Ozcan et al.,
2006; Kandula and Agarwal, 2011). The significant overlap between
the side effects of VEGF inhibitors and the features of pre-eclampsia
provides strong circumstantial evidence of the importance of VEGF in
pre-eclamptic pathophysiology. This rationale has been confirmed
in vivo, where sFLT-1 administration led to the development of hyper-
tension in rodent models (Maynard et al., 2003; Kumasawa et al.,
2011). This is thought to be due to reduced nitric oxide and prostacyc-
lin production, both of which are regulated by VEGF (He et al., 1999a;
Venkatesha et al., 2006).
It can now be appreciated how excessive production of sFLT-1, as
seen in pre-eclampsia, acts as a VEGF trap to cause the clinical features
of disease. Through reducing VEGF bioavailability, sFLT-1 negatively
impacts on both microvascular tone and endothelial cells within the
glomerulus, causing hypertension and proteinuria.
The many variants of sFLT-1
sFLT-1 results either from alternative splicing of the FLT-1 pre-mRNA
(He et al., 1999b) or through cleavage of the ectodomain of FLT-1
(Rahimi et al., 2009). Proteolytic cleavage of the extracellular region is
thought to occur adjacent to the transmembrane domain (Raikwar
et al., 2016) and is potentially due to the actions of proteolytic enzymes,
such as ADAM10, ADAM17 or MMP-14 (Raikwar et al., 2014; Han
et al., 2015). Proteolytic cleavage produces an sFLT-1 that is identical to
the extracellular region of FLT-1, while the multiple variants of sFLT-1
that have been identified all have unique C-terminal sequences (Sela
71
et al., 2008; Heydarian et al., 2009; Thomas et al., 2009). The signifi-
cance of these different splice variants currently remains unknown.
Alternative splicing has been found to occur in >80% of genes in
the human genome with the majority of splicing events affecting the
coding sequence (Matlin et al., 2005). Splicing events are regulated by
cis- and trans-elements. The former are sequences within the pre-
mRNA that help to direct splicing, such as exon splicing enhancers or
silencers (ESEs or ESSs, respectively) and their intronic counterparts
(ISEs and ISSs), as well as polyadenylation signals within the sequence.
Trans-elements are cellular factors such as RNA or protein that regu-
late splicing. For example, the SR family of RNA-binding proteins bind
to ESEs and assist in the assembly of the spliceosome complex (Matlin
et al., 2005).
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The FLT-1 gene encodes an mRNA transcript containing 30 exons.
The FLT-1 pre-mRNA has been shown to give rise to the full-length
membrane-bound FLT-1, as well as four truncated soluble splice var-
iants (Fig. 1). Of these, three sFLT-1 variants appear to be translated
into protein, two of which are up-regulated in pre-eclamptic placenta
(Heydarian et al., 2009). The two variants altered in the setting of pre-
eclampsia are sFLT-1 i13 (also known as sFLT-1; sFLT-1_v1
(Heydarian et al., 2009)) and sFLT-1 e15a (also known as sFLT-1_v2
(Heydarian et al., 2009); sFLT-1 14 (Sela et al., 2008)). As shown in
Fig. 1, these splice variants share significant sequence homology with
the full-length FLT-1 receptor, differing only at their C-terminus.
sFLT-1 i13 was the first sFLT-1 identified (Kendall and Thomas,
1993) and comprises the first 13 exons of FLT-1 with an extension of
exon 13 into the intronic sequence due to a read through of the exon
13–14 splice site (Heydarian et al., 2009; Thomas et al., 2009). Intron 13
contains two widely separated polyadenylation signal sequences that
are cis-elements regulating the alternative splicing of the i13 variant.
This produces either a short or long 3′-untranslated region (UTR)
depending on which polyadenylation sequence is utilized (Thomas
et al., 2007). The impact of alterations in the 3′-UTR is currently
unknown, but has been suggested to possibly regulate mRNA expres-
sion in a tissue-specific or temporal fashion, or influence mRNA stabil-
ity or its translational efficiency (Thomas et al., 2007). As a result, after
657 amino acids, the i13 protein diverges from full-length FLT-1,
encoding a unique 31 amino acid C-terminal tail and producing an 85–
95 kDa protein.
In comparison, sFLT-1 e15a was identified more recently (Thomas
et al., 2007). Interestingly, it appears this variant is specific to humans
and higher order primates, having resulted from the insertion of an
AluSq sequence into the primate genome following a retrotransposi-
tion event ~40 million years ago (Thomas et al., 2009). Alu sequences
are common and represent 6–13% of human genomic DNA (Mighell
et al., 1997). They are commonly found within intronic and non-coding
DNA; however, they are capable of affecting gene transcription. They
contain many stop codons and can also affect splicing to produce trun-
cated proteins. Through their impact on splicing, they have been linked
to a number of human diseases, such as neurofibromatosis, Alport
syndrome and familial hypercholesterolaemia (Mighell et al., 1997).
Similarly, the introduction of the AluSq sequence leading to alternative
sFLT-1 splicing could have contributed to the appearance of pre-
eclampsia, a disease that is unique to humans and perhaps some higher
order primates (Thornton and Onwude, 1992).
sFLT-1 e15a arises from the use of a previously unknown splice
acceptor site within intron 14 producing a protein consisting of the first
72 Palmer et al.

Extracellular domain Transmembrane domain Intracellular domain

Flt-1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

sFlt-1 1 2 3 4 5 6 7 8 9 10 11 12 13

sFlt-1 i13 13 i13

sFlt-1 e15a 13 14 15a

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sFlt-1 v3 13 14 15b

sFlt-1 v4 13 14 i14

Figure 1 Schematic representations of FLT-1 splice variants. The FLT-1 gene encodes the full-length membrane-bound FLT-1 receptor VEGFR-1, as
well as the four soluble alternative sFLT-1 splice variants. All transcripts are identical to exon 13, with the soluble transcripts each identified by a unique
C-terminal region. Of these, sFLT-1 i13, sFLT-1 e15a and sFLT-1 v4 are translated into protein with only the former two being increased in pre-
eclampsia. Reprinted with permissions from Palmer et al. (2015).

14 exons of FLT-1 followed by a 480 nucleotide stretch of intronic
sequence (Sela et al., 2008). This encodes the unique alternatively
spliced exon 15a followed by a 3′-UTR containing the complete AluSq
sequence. A polyadenylation signal sequence inserted as a result of the
Alu sequence likely directs the alternative splicing of exon 15a, which
encodes a unique polyserine tail (Thomas et al., 2007). The resulting
protein shares 706 amino acids with FLT-1 and has a unique 28 amino
acid tail, producing a protein that is 95–135 kDa in size due to varia-
tions in the level of glycosylation (Gu et al., 2008). Importantly, sFLT-1
e15a is absent from all mammals except humans and higher order pri-
mates, highlighting it as potentially the key variant underlying pre-
eclamptic pathophysiology.
sFLT-1 splice variant mRNA
expression and regulation
Both sFLT-1 i13 and sFLT-1 e15a are capable of binding to VEGF and
antagonizing its actions (Kendall et al., 1994; Sela et al., 2008; Palmer
et al., 2015). The i13 variant is also known to antagonize PlGF (Kendall
et al., 1994) and it is likely the e15a variant does too, given the preser-
vation of the ligand-binding site. Interestingly, the e15a variant is the
predominant isoform in human placenta compared to i13, which is the
main variant in rhesus monkey placenta (Thomas et al., 2009).
Furthermore, in humans sFLT-1 e15a appears to be almost exclusively
expressed by the placenta where its expression is ≥600-fold higher
than in the other organ systems where it is found (Jebbink et al., 2011).
Certainly within the placenta, sFLT-1 e15a is the predominant isoform
of FLT-1 mRNA, constituting >80% of transcripts compared to sFLT-1
i13, which constitutes 12%, and membrane-bound FLT-1, which is
<5% (Jebbink et al., 2011). There are many extra-placental sources of
sFLT-1 (He et al., 1999b; Jebbink et al., 2011), which are likely to
mainly be the i13 isoform as it is widely expressed throughout the
body (Rajakumar et al., 2005; Suwaki et al., 2006; Souders et al.,
2015). However, with the exception of human brain microvascular
endothelial cells, it is always expressed to a lesser extent than the
membrane-bound FLT-1 (Jebbink et al., 2011). While this suggests a
tissue-specific expression profile of FLT-1 transcripts, expression also
appears to be cell-type specific. In blood vessels, endothelial cells
appear to exclusively produce the i13 variant, whereas the underlying
vascular smooth muscle exclusively expresses the e15a variant (Sela
et al., 2008). Furthermore, there is the potential for temporal regula-
tion of expression, as splicing patterns have been shown to alter over
the course of pregnancy in mice. In murine placenta, Flt-1 predomi-
nates early in gestation before sFlt-1 becomes more highly expressed
from the second trimester onwards (He et al., 1999b). Whether this
holds true in humans remains to be fully characterized; however, it is
possible given the rise in circulating sFLT-1 seen with advancing gesta-
tion (Levine et al., 2004; Palmer et al., 2015).
Within the placenta, sFLT-1 e15a is mainly produced by the syncy-
tiotrophoblast and cytotrophoblast, where its mRNA expression is
100-fold greater than in placental vascular smooth muscle cells, endo-
thelial cells and macrophages (Thomas et al., 2009). In the setting of
pre-eclampsia, all FLT-1 transcripts appear up-regulated (Jebbink et al.,
2011); however, sFLT-1 e15a shows the greatest increase, with levels
appearing to correlate with severity of pre-eclampsia (Whitehead
et al., 2011).
Placental hypoxia is a key factor in pre-eclamptic pathophysiology.
Many transcriptional regulators have been shown to be involved in
increasing the expression of both FLT-1 and sFLT-1 in response to hyp-
oxia, such as hypoxia inducible factor (Gerber et al., 1997; Nagamatsu
et al., 2004; Tal et al., 2010), growth arrest and DNA damage 45
(Xiong et al., 2009) and early growth response protein 1 (Vidal et al.,
2000). Similarly, many key molecules in the hypoxic response also
increase the expression of sFLT-1, such as VEGF (Gerber et al., 1997)
and adenosine through the up-regulation of a key enzyme, CD73
Placental-specific sFLT-1 73

(Iriyama et al., 2015). Furthermore, the mechanism controlling the
regulation of alternative splicing of FLT-1 also appears to be hypoxia
dependent. Recently, it has been shown that a nuclear protein, known
as jumonji domain containing protein-6 (JMJD6) is a regulator of alter-
native splicing of the FLT-1 pre-mRNA (Boeckel et al., 2011). Under
normoxic cellular conditions, JMJD6 is able to interact with a compo-
nent of the spliceosome complex, U2AF65, hydroxylating it.
Hydroxylated U2AF65 then directs the splicing machinery to produce
full-length FLT-1. Importantly, they were able to show that under hyp-
oxic conditions, there was inhibition of the interaction of JMJD6 and
U2AF65, as JMJD6 requires oxygen for its enzymatic function. Under
these conditions, within endothelial cells, non-hydroxylated U2AF65
directed splicing to produce sFLT-1 (Boeckel et al., 2011). We have
expression, as outlined in Table I. Interestingly, in vitro treatment with
both YC-1 and pravastatin led to a significant reduction solely in sFLT-1
e15a mRNA expression with no significant effect seen on the sFLT-1
i13 variant (Brownfoot et al., 2015a,b). Metformin led to a significant
reduction in sFLT-1 i13 mRNA expression in endothelial cells and sFLT-1
e15a mRNA expression from cytotrophoblast and placental explants
(Brownfoot et al., 2016), while treatment with ouabain led to a signifi-
cant reduction in both sFLT-1 i13 and e15a variant expression in pla-
cental explants, though with a trend towards greater repression of the
e15a variant (Rana et al., 2014). These findings all support the import-
ance of sFLT-1 in pre-eclampsia and potentially that targeting therapies
that reduce the placental-specific variant is crucial for effectively redu-
cing circulating sFLT-1 in early-onset pre-eclampsia.

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recently shown this mechanism of FLT-1 splicing also occurs within the
placenta, where the silencing of JMJD6 expression or the effects of
hypoxia led to an up-regulation of both sFLT-1 i13 and sFLT-1 e15a
(Palmer et al., 2016b). Importantly, it was also noted that the expres-
sion of JMJD6 is significantly reduced in the pre-eclamptic placenta,
likely secondary to the effects of hypoxia (Palmer et al., 2016b).
With an increased understanding of the pathophysiology of pre-
eclampsia, many are now actively seeking new therapies that could
provide an alternative to delivery. A number of drugs under investiga-
tion have also explored their effect on sFLT-1 variant mRNA
The impact of circulating sFLT-1
e15a
While many studies have assessed altered mRNA expression of the
sFLT-1 splice variants, limited work has been undertaken on the pro-
tein due to a lack of any commercially available assay. Furthermore, as
detailed above, each splice variant has a very small unique tail against
which antibodies can be developed to ensure specificity for the

Table I Effects of potential pre-eclamptic therapeutics on sFLT-1 variant mRNA expression level in vitro.

Drug Mechanism of action Cells treated Culture Length of [Drug] sFLT-1 i13* sFLT-1
(human) conditions treatment e15a*
(hours)
.............................................................................................................................................................................................
Metformin Inhibition of complex I in the HUVEC 10 ng/ml TNF-α, 24 2 mM <0.05 N/A
(Brownfoot et al., mitochondrial transport chain 20% O2 5 mM <0.01
2016)
Villous CTB 8% O2 48 2 mM N/A <0.00001
5 mM <0.00001
Placental explants 8% O2 72 2 mM N/A <0.05
5 mM <0.0001

Ouabain (Rana et al., Inhibition of HIF-1α through Normal and PET 21% O2 72 500 nM <0.05 <0.05
2014) phosphorylation of HSP27 placental explants

YC-1 (Brownfoot Induces NO, activates Primary trophoblast 8% O2 24 1 μM NS NS

et al., 2015b) guanylyl cyclase and inhibits 10 μM NS NS
HIF-1α
100 μM NS <0.001
PET placental 8% O2 72 1 μM NS NS
explants 10 μM NS NS
100 μM NS <0.0001

Pravastatin Inhibiting the HMG-CoA HUVEC 1% FCS and 6 200 μmol/l NS <0.001
(Brownfoot et al., reductase pathway 10 ng/ml TNF-α 2000 μmol/l NS <0.0001
2015a)
Early-onset PET 20% O2 48 2000 μmol/l NS <0.05
placental explants
Pre-/post-delivery- 20% O2 48 200 μmol/l NS NS
treated explants 2000 μmol/l NS <0.0001

[Drug], drug concentration; TNF, tumour necrosis factor; NS, not significant; N/A, not available; HIF-1α, hypoxia inducible factor 1α; HSP27, heat-shock protein 27; NO, nitric oxide;
PET, pre-eclampsia; FCS, foetal calf serum; HMG-CoA, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase and CTB, cytotrophoblast. All cells tested were cultured under standard
conditions at 37°C in 5% CO2 with oxygen conditions as stated.
*All statistically significant changes refer to a decrease in sFLT-1 variant mRNA expression in experimental groups compared to control treated cells. A t-test was used for statistical
analysis in all studies.
74 Palmer et al.

particular sFLT-1 splice variant of interest. These antibodies, devel-
oped in the non-commercial setting, enable the assessment of how the
translated sFLT-1 proteins function and their expression in both health
and disease (see Fig. 2).
We have developed an ELISA that specifically detects the sFLT-1
e15a variant (Palmer et al., 2015). This involved developing a peptide
sequence from the unique tail of the variant protein (Fig. 2; boxed
sequence). This peptide was then coupled to a highly immunogenic
protein for animal immunization. Once an appropriate antibody
response had occurred, polyclonal antibodies were isolated from the
serum and purified for testing. Importantly, both the antibody and the
subsequent sFLT-1 e15a ELISA that was developed are specific to this
isoform. Importantly, an independent group has also successfully devel-
conditions of placental malfunction, such as foetal growth restriction
(Palmer et al., 2016a), although this requires further investigation.
Implications for clinical practice
The discovery of multiple sFLT-1 splice variants provides the potential
to better identify disorders of placentation through targeting strategies
towards the placental-specific variant, sFLT-1 e15a.
The use of both anti-angiogenic and angiogenic factors as biomar-
kers for the prediction or diagnosis of pre-eclampsia was identified as
soon as their importance in the disease process was realized (Hertig
et al., 2004). Subsequent work has shown that total sFLT-1, particu-
larly as a ratio with PlGF, appears able to diagnose and even predict

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oped monoclonal antibodies targeting both sFLT-1 i13 and a peptide
sequence seen in FLT-1, sFLT-1 e15a, sFLT-1_v3 and sFLT-1_v4
(Souders et al., 2015) (Fig. 2; underlined sequences). As it is used on
serum, Souders et al. believe it will be relatively specific for sFLT-1 e15a
due to the membrane-bound nature of FLT-1 and the exceptionally
low expression of the other sFLT-1 splice variants. However, cleavage
of FLT-1 to produce a cleaved soluble version is also possible, which
may impact on the specificity of this antibody (Raikwar et al., 2014).
Using these novel systems, both groups have shown that sFLT-1 e15a
gradually rises with advancing gestation (Palmer et al., 2015; Souders
et al., 2015) and is significantly elevated in women who go on to develop
pre-eclampsia and in those diagnosed with the early-onset form (Palmer
et al., 2015, 2016a; Souders et al., 2015). Interestingly, the sFLT-1 i13
variant is the one most significantly increased in women who develop
pre-eclampsia >34 weeks gestation (Souders et al., 2015; Palmer et al.,
2016a), perhaps due to widespread maternal endothelial dysfunction,
thought to be the main contributor to late-onset pre-eclampsia.
Encouragingly, we have also determined that the sFLT-1 e15a vari-
ant protein is biologically active, being capable of antagonizing the
actions of VEGF (Palmer et al., 2015). Endothelial cell function is also
impacted by the sFLT-1 splice variants, with evidence of impaired
endothelial cell invasion, migration and tubule formation in the pres-
ence of either sFLT-1 i13 or sFLT-1 e15a (Palmer et al., 2015).
Importantly, normal VEGF signalling and endothelial function were
maintained with the addition of heat inactivated sFLT-1 e15a (Palmer
et al., 2015). Furthermore, the over-expression of the sFLT-1 e15a
variant in mice produces a pre-eclamptic phenotype, regardless
of whether an adenoviral (Szalai et al., 2014, 2015) or lentiviral
(Kumasawa et al., 2011) expression system is used. In combination,
these works confirm the ability of the sFLT-1 e15a protein to cause
endothelial dysfunction and the clinical features of pre-eclampsia. It
is also possible that sFLT-1 e15a expression is altered in other
the development of early-onset pre-eclampsia (Schiettecatte et al.,
2010; Benton et al., 2011; Verlohren et al., 2012; Villa et al., 2013; Liu
et al., 2015; Andersen et al., 2016; Zeisler et al., 2016). However, the
diagnostic accuracy of this test for late-onset pre-eclampsia remains
poor. This test is yet to be widely integrated into clinical practice and
recent work shows that the test performs better as a negative pre-
dictor of early-onset pre-eclampsia rather than being able to accur-
ately predict who will develop the disease (Zeisler et al., 2016).
As these tests detect total sFLT-1, their accuracy may be hampered
by the presence of both cleaved and alternatively spliced sFLT-1,
particularly sFLT-1 i13 that has a strong endothelial source. For
example, accurate diagnosis of early-onset pre-eclampsia may be lim-
ited when using total sFLT-1 levels in women with pre-existing endo-
thelial dysfunction (such as obesity and diabetes) who do not have
pre-eclampsia due to increased endothelial secretion of sFLT-1 i13.
Therefore, developing a test that solely detects the placental-specific
sFLT-1 variant, sFLT-1 e15a, may have improved accuracy in disorders
of placental function, such as early-onset pre-eclampsia. Furthermore,
the ability to detect the different splice variants of sFLT-1 may also
provide better accuracy for detecting term pre-eclampsia. As term
pre-eclampsia likely results from underlying maternal endothelial dys-
function rather than placental disease, a test that can distinguish
between the cellular origins of sFLT-1 may be more accurate. Here,
potentially a ratio of sFLT-1 e15a to sFLT-1 i13 may be useful. For
example, in early-onset pre-eclampsia, the ratio would be higher (e15a
> i13), because the e15a variant is likely to have greater expression
than the i13 variant. In comparison, in late-onset pre-eclampsia, the
ratio would be lower (e15a < i13), as widespread endothelial dysfunc-
tion is likely to cause a greater expression of the i13 variant to the
e15a variant. Preliminary data support this supposition, where no sig-
nificant increase in sFLT-1 e15a is seen in term disease, while total
sFLT-1 is significantly increased (Palmer et al., 2016a). Moreover, this

FLT-1 650 kkeirdqeapyllrnlsdhtvaisssldchangvpepqitwnnhkiqqepgiilgpgsstlfiervtee…

sFLT-1 i13 650 kkeirgehcnkkavfsriskstrndcqsnvkh
sFLT-1 e15a 650 kkeirdqeapyllrnlsdhtvaisssldchangvpepqitwnnhkiqqepelytstspsssssspls[11]

Figure 2 Alignment of the amino acid sequence for human FLT-1, sFLT-1 i13 and sFLT-1 e15a. The amino acid sequence for all proteins is shown
from amino acid 650: sequence prior to amino acid 650 shares 100% homology between the three proteins. The unique C-terminal tails are highlighted
for sFLT-1 i13 and sFLT-1 e15a (grey). Antibodies that have been developed are indicated with the underlined sequences indicating those used by
Souders et al. (2015) and the boxed sequence by Palmer et al. (2015). The sFLT-1 e15a polyserine tail continues for a further 11 serine residues.
Placental-specific sFLT-1
premise is strongly supported by the fact that those developing term
pre-eclampsia have an average 3-fold increase in circulating sFLT-1
levels compared to normal term controls, whereas a 43-fold increase
in serum sFLT-1 is seen in early-onset disease compared to preterm
controls (Wikstrom et al., 2007). However, this remains an area
requiring further investigation as studies performed to date on late-
onset pre-eclampsia were limited by a small sample size. Furthermore,
research in this area is currently limited by the absence of any com-
mercially available assays.
Discussion
Pre-eclampsia remains a significant burden to women and their health-
care providers globally, being directly responsible for 14% of maternal
deaths globally (Say et al., 2014). While the bulk of the maternal mortal-
ity is sadly in developing countries, pre-eclampsia remains a major cause
of maternal mortality even in the developed world (Johnson et al.,
2014). The rapid advancement in our understanding of how pre-
eclampsia arises, however, offers hope through aiding the development
of new approaches to improve how we predict, diagnose and treat it.
The discovery of multiple different splice variants of sFLT-1 has led
to the finding of a placental-specific variant, sFLT-1 e15a (Sela et al.,
2008; Heydarian et al., 2009; Thomas et al., 2009). This splice variant
is seen only in humans and is principally expressed in the placenta
(Jebbink et al., 2011), making it likely to be the variant chiefly respon-
sible for the clinical features of early-onset pre-eclampsia.
Importantly, the sFLT-1 e15a variant has been shown to be biologic-
ally active and capable of producing endothelial dysfunction (Palmer
et al., 2015). Furthermore, sFLT-1 e15a is significantly elevated in
women with preterm pre-eclampsia (<34 weeks gestation), with levels
elevated prior to the onset of clinical symptoms (Palmer et al., 2015,
2016a; Souders et al., 2015). It is also significantly reduced by a num-
ber of therapeutic agents that show promise as new drugs with which
to treat pre-eclampsia (Rana et al., 2014; Brownfoot et al., 2015a, b,
2016).
The sFLT-1 variants, with their differing tissue specificity, therefore
offer a unique opportunity to capitalize on the potential of sFLT-1 as a
predictive or diagnostic marker of pre-eclampsia. Development of
accurate assays to measure these variants may significantly improve test
performance (both in combination with PlGF or alone) for both early
and late-onset disease. The development of an accurate test could sig-
nificantly advance the delivery of obstetric care globally without increas-
ing the economic burden on already financially challenged healthcare
systems (Vatish et al., 2016). Furthermore, in better understanding the
pathways involved in the release of anti-angiogenic factors from the pla-
centa and the role the different splice variants play, it may assist in
improved therapeutic design and assessment of drug effectiveness.
Authors’ roles
K.R.P. developed the concept, undertook the literature review and
wrote the manuscript. S.T. contributed to concept design. Both S.T.
and T.J.K. provided draft revisions and final approval for publication.
Funding
Funded by National Health and Medical Research Council (NHMRC)
grants (#1101871, #1061977). NHMRC also provided salary support
75
(#1050765 to S.T. #1062418 to T.J.K.). K.R.P. received salary support
from the Department of Obstetrics and Gynaecology, Monash
University.
Conflict of interest
None declared.
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