Recent Advances in Understanding of Preeclampsia

REVIEW
Recent Advances in Understanding of Preeclampsia
Yuval Bdolah1, S. Ananth Karumanchi1,2, Benjamin P. Sachs1
Departments of Obstetrics and Gynecology1 and Medicine2, Beth Israel Deaconess Medical
Center, Harvard Medical School, Boston, Mass, USA
2005;46(5):728-736
J
Abstract
Despite intensive research, preeclampsia still accounts for significant morbidity and mortality for the
ed
mother and the neonate, especially in developing countries. Recent studies have suggested that excess
M
secretion of a naturally occurring anti-angiogenic molecule of placental origin referred to as soluble
t
fms-like tyrosine kinase-1 (sFlt-1, also referred to as sVEGFR-1) may contribute to the pathogenesis of
oa
preeclampsia. sFlt-1 acts by antagonizing two pro-angiogenic molecules – vascular endothelial growth
Cr
factor (VEGF) and placental growth factor (PlGF). Abnormalities in the angiogenic balance have been
proposed as having a major role in the molecular cascade leading to proteinuria, hypertension, and en-
dothelial dysfunction. Further evidence supports the hypothesis that angiogenic balance is crucial to
differentiation and invasion of cytotrophoblasts. The abnormal placentation and the accompanying
hypoxia may, in turn, result in more sFlt-1 production, thus leading to a vicious cycle of sFlt-1 produc-
tion, eventually causing preeclampsia. These recent discoveries may facilitate the development of
novel strategies for the diagnosis and therapy of preeclampsia.
Despite intensive research, preeclamp-
immunogenetics, have been suggested as poten-
sia still accounts for significant morbidity and mor-
tial “preeclampsia-genes” (7). Due to familial clus-
tality for the mother and the neonate, complicat-
tering of the disease, some chromosomal loci have
ing 5-7% of all pregnancies and exposing them to
also been implicated as being linked to pre-
a 3- to 25-fold increased risk of severe obstetric
eclampsia (4,5,10). Two recent Dutch studies
complications (1-3). Recent advances in the un-
identified another genetic locus that is signifi-
derstanding of the pathophysiology of preeclamp-
cantly linked with preeclampsia on 10q22 (14,15).
sia have focused renewed attention on preeclam-
Yet, no single gene or chromosomal locus cur-
psia research.
rently known can explain the complex patho-
genesis of preeclampsia. One protein that has re-
The etiologic factors causing this disease
cently been implicated by our group as being able
are still not completely clear, although evidence
to reproduce some of the major signs of pre-
supports involvement of genetic, immune, angio-
eclampsia in rodents is soluble fms-like tyrosine
genic, and other mechanisms. Preeclampsia is
kinase-1 (sFlt-1) (16). sFlt-1 is a splice variant of
long believed to be related to one or more genes
vascular endothelial growth factor (VEGF) recep-
and the search for genetic factors predisposing to
tor 1 that is produced in excess quantities by the
preeclampsia has been intense (4-10). The familial
preeclamptic placentas. When injected to preg-
factor in preeclampsia was noted long ago (11)
nant rats exogenous sFlt-1 caused hypertension,
and since then genes involved in hemodynamic
proteinuria, and glomerular endotheliosis, the
changes of pregnancy (12,13) in thrombophilia,
hallmarks of preeclampsia. This finding has led
and in oxidative stress, and genes involved in
many studies to focus on the angiogenesis profile
728
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Croat Med J
2005;46(5):728-736
in the preeclamptic placenta, and in the serum and
der to form an uteroplacental circulation. The first
urine of preeclamptic patients (17-24). It also
chorionic villi vessels are a primitive vascular net-
caused a renewed interest in the role of hypoxia in
work that arises through a primary process termed
preeclampsia and stimulated studies unraveling
vasculogenesis (29,30). Later on, the secondary
Bdolah
the link between angiogenesis and hypoxia in the
stage of angiogenesis takes place, forming new
placental trophoblasts (25-27).
vessels in a non-branching fashion. The process of
et
This review will focus on recent ad-
trophoblast invasion and remodeling of the spiral
al:
vances in the emerging field of placental angio-
arteries that has been studied extensively by Fisher
A
genesis and its association with hypoxic condi-
at al (31-35) seems to have an utmost significance
dvances
tions, as well as describe some of the potential di-
to the success of a pregnancy and when defective,
rections of future research.
preeclampsia most likely follows. During this pro-
cess, a subset of the trophoblast cells, the extra-
in
Key Players in Placental
villous trophoblasts, transform to endothelial phe-
Preeclampsia
Angiogenesis in Health and
notype cells, thus expressing typical endothelial
Disease
markers such as vascular endothelial-cadherin
Adequate placentation involves a two
(VE-cadherin), and a b integrin (33). Failure of
V 3
stage mechanism (28). Trophoblast invasion re-
this transformation most likely results in an inade-
quires a process of vascularization to establish a
quate blood supply to the growing placenta,
fetoplacental vascular network, and finally, com-
hypoxia, and a shift in the secretion of angiogenic
plete remodeling of the spiral arteries ensues in or-
factors (Fig. 1). Preeclampsia has recently been de-
Figure 1. Abnormal placentation in preeclampsia. Exchange of oxygen, nutrients, and waste products between the fetus and
the mother depends on adequate placental perfusion by maternal vessels. In normal placental development, invasive
cytotrophoblasts of fetal origin invade the maternal spiral arteries, transforming them from small-caliber resistance vessels
to high-caliber capacitance vessels capable of providing placental perfusion adequate to sustain the growing fetus. During
the process of vascular invasion, the cytotrophoblasts differentiate from an epithelial phenotype to an endothelial pheno-
type, a process referred to as “pseudovasculogenesis.” In preeclampsia, cytotrophoblasts fail to adopt an invasive endothe-
lial phenotype. Instead, invasion of the spiral arteries is shallow and they remain small caliber, resistance vessels. This may
result in the placental ischemia.
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Croat Med J
2005;46(5):728-736
scribed as a state of imbalance between pro-
preeclampsia were associated with decreased cir-
angiogenic and anti-angiogenic factors (18). The
culating levels of free VEGF and PlGF. Hence, a
main pro-angiogenic factors that promote angio-
logical assumption was that excess circulating
genesis in the placenta belong to the VEGF family.
sFlt-1 may lead to an anti-angiogenic state and
In addition to the VEGF family members, angio-
cause endothelial dysfunction and the clinical syn-
poietins (Ang-1 and Ang-2) and their receptor
drome of preeclampsia.
Tie-2 are also expressed abundantly in the normal
A few additional experiments further es-
placenta (36-39) and act in the later stages of
Preeclampsia
tablished the role of sFlt-1 as a key anti-angiogenic
in
angiogenesis.
molecule, predominantly involved in the patho-
VEGF-A, the main family member of the
physiology of preeclampsia. First, we found that
VEGF family in the placenta, binds to two tyrosine
preeclamptic serum inhibited endothelial tube for-
dvances
kinase receptors, Flt-1 (also referred to as VEGFR-
mation, an effect that disappeared once repeated
A
1) and kinase insert domain receptor (KDR) (hu-
with serum from 48 hours post-partum. These re-
al:
man) /Flk-1 (murine), (also known as VEGFR-2)
sults could be reproduced when sFlt-1 was added
et
(40). Other than VEGF, the other major pro-angio-
to normotensive serum at concentrations noted in
genic protein is placental growth factor (PlGF).
patients with preeclampsia, and could be restored
VEGF-A and PlGF are produced by almost all types
Bdolah
by adding exogenous VEGF and PlGF (16). This
of trophoblasts (22). Both VEGF receptors Flt-1 and
study suggested to us that the anti-angiogenic
KDR are expressed on trophoblast cells in addition
properties of serum from preeclamptic patients
to endothelial cells.
were due to blockade of VEGF and PlGF by excess
sFlt-1 is the major endogenous inhibitor
circulating sFlt-1. Finally, gene transfer of sFlt-1
of angiogenesis found in the placenta. This potent
into pregnant rats by an adenoviral vector pro-
antiangiogenic protein is encoded by alternative
duced hypertension, proteinuria, and glomerular
splicing of the Flt-1 gene, leading to a shorter
endotheliosis, the classical pathological renal le-
extracellular domain that still retains the ability to
sion of preeclampsia (16). Hence, we concluded
bind to VEGF and PlGF. Thus, when circulating in
that excess sFlt-1 made by preeclamptic placentas
the serum, sFlt-1 can bind to VEGF and PlGF and
might be responsible for the hypertension and
prevent them from binding to cell-surface recep-
proteinuria of preeclampsia by inducing a defi-
tors. Clark et al (41) have shown by in situ hybrid-
ciency of VEGF and PlGF.
ization that trophoblasts express the sFlt-1 messen-
Recently, a case-control study using
ger RNA. Serum from pregnant women has been
blood samples from the Calcium for preeclampsia
found to contain a VEGF-binding protein that later
Prevention trial (CPEP) was performed, measuring
was confirmed to be sFlt-1 (42,43).
circulating angiogenic markers in patients with
The hypothesis that the preeclamptic
preeclampsia and matched controls in order to de-
placenta elaborates soluble factors, which induce
termine if changes in their levels antedate the clin-
endothelial cell dysfunction was first suggested by
ical symptoms and signs of preeclampsia (17).
Roberts et al (44,45). After sFlt-1 was suggested as
During the third trimester of pregnancy, the level
a candidate molecule that induces maternal
of sFlt-1 increased and the level of PlGF decreased
preeclampsia (16), several studies have confirmed
in healthy controls, an effect that was very pro-
high levels of circulating sFlt-1 in maternal serum
nounced in preeclampsia patients (17). The sFlt-1
(16,17,20-24).
level started increasing five weeks before the onset
sFlt-1 was initially identified as a poten-
of preeclampsia, and was accompanied by de-
tial soluble factor that mediates maternal endothe-
creases in both free PlGF and VEGF. Other groups
lial dysfunction by gene expression profiling of
have reported similar changes in PlGF during the
placental tissue from women with and without
second trimester in women destined to develop
preeclampsia. Using microarray chips mRNA for
preeclampsia (23,46). The decreases in PlGF oc-
sFlt-1 was found to be dramatically up-regulated in
curred even as early as the first trimester (although
preeclamptic placentas (16). In addition, sFlt-1 lev-
not as dramatically as in the second trimester),
els in patients with preeclampsia were found to fall
which has also been used in other studies as a pos-
to baseline 48 hours after delivery. Increased cir-
sible prediction tool for the early diagnosis of
culating sFlt-1 concentrations in patients with
preeclampsia (47-49).
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2005;46(5):728-736
A most recent study (19) measured uri-
Endothelial and tumor mammalian cells
nary concentrations of PlGF in archived urine sam-
are known to express proteins essential for inva-
ples from the CPEP study. The same cohort of
sion in hypoxic conditions. These up-regulate the
women used for the previous trial (17) was used in
expression of heat shock and glucose-regulated
Bdolah
this study. The results suggested that urinary con-
proteins, as well as cytokines and growth factors
centrations of PlGF were significantly lower at
(55-59). Such proteins are, for example, endothe-
mid-gestation among women who subsequently
lin (60), vascular endothelial growth factor (VEGF)
et
al:
developed preeclampsia. These results were re-
(61) and IL-1a (62). Landmark studies showed that
produced in a further study (50). Together, all
hypoxia could induce expression of platelet de-
A
dvances
these data imply that sFlt-1 binds to PlGF and
rived growth factor (PDGF) mRNA (63) and VEGF
VEGF, thereby, causing a shift towards an anti-
mRNA (61) in tissue culture, indicating that oxy-
angiogenic state, which in turn causes maternal
gen was an important regulator of angiogenesis. A
in
endothelial damage and clinical preeclampsia.
large number of genes involved in different steps
Preeclampsia
of angiogenesis such as angiopoietins, fibroblast
growth factors and their various receptors, and
Placental Hypoxia: Cause or
genes involved in matrix metabolism are inde-
Effect?
pendently responsive to hypoxia in tissue culture.
It has been known for some years that in
Rajakumar and Conrad (64) investigated
preeclampsia, endovascular invasion of cytotro-
the expression of hypoxia-inducible factor (HIF) in
phoblasts remains superficial and the uterine
normal human placentas and found HIF-1a and
blood vessels do not undergo adequate vascular
-2a mRNA present in placentas of all gestational
transformation. The spiral arteries fail to convert
ages. Both were expressed by the syncytiotro-
from small caliber vessels to large capacitance
phoblast, villous cytotrophoblast, and fetoplacen-
uteroplacental arteries (51). Furthermore, Zhou et
tal vasculature. In two additional studies (65,66)
al (32) have shown that the invasive trophoblasts
they demonstrated that the protein expression of
fails to undergo the process of pseudo-vasculo-
HIF-2a, but not of HIF-1a or -1b, is selectively in-
genesis. The functional consequences of these ab-
creased in the preeclamptic placenta and is not
normalities are still unknown, but it is likely that
down-regulated upon oxygenation. As both Flt-1
there is compromise of blood flow into the inter-
and VEGF are hypoxia-inducible genes (67,68), re-
villous space leading to placental ischemia. Addi-
sponding to HIF and are induced in preeclamptic
tionally, it has been claimed that in preeclampsia,
placentas, these data may support a different
placental ischemia is an early event (52), leading
mechanism of induction, in the face of the un-
to placental production of soluble factors that
changing HIF-1a in preeclampsia. Furthermore,
cause maternal endothelial dysfunction, and re-
until recently it was unclear what the net effect of
sulting in the clinical disease. These hypotheses
placental hypoxia would be on the balance of Flt-1
have led to a fundamental question: Is placental
and VEGF expression (as both are hypoxia-induc-
hypoxia in preeclampsia a cause or an effect of sol-
ible). Nagamatsu et al (25) have demonstrated that
uble factors, such as sFlt-1 (26)?
lowering the oxygen percentage from 20% O to
2
Reduction in the placental perfusion
8% O and to 2% O in a primary cytotrophoblast
2
2
pressure has long been thought to underlie
culture caused a rise in sFlt-1 concentration and
preeclampsia. Uteroplacental ischemia as an ani-
sFlt-1mRNA was strikingly increased. Although to-
mal model of preeclampsia has been extensively
tal VEGF levels in these cells increased modestly
studied, based on the fact that in women destined
with hypoxia, free VEGF levels were undetectable
to develop preeclampsia, uteroplacental blood
along with very low free PlGF concentrations in
flow is reduced by 50-70% (53). As early as 1939,
the media in the presence of stable PlGF mRNA
Ogden et al (54) tried to clamp the descending
levels. This study provided some evidence that ex-
aorta of anesthetized dogs and induced about a
cess sFlt-1 production seen in preeclampsia may
50% reduction in placental perfusion pressure.
be a consequence of placental hypoxia. However,
Pregnant dogs’ blood pressure increased about 25
it does not rule out the possibility that alterations
mm immediately. This effect could not be demon-
in placental sFlt-1 in preeclampsia may be primary
strated in non-pregnant dogs.
and directly lead to the abnormal placentation/pla-
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Croat Med J
2005;46(5):728-736
cental hypoxia. Ahmad and Ahmed (27) have also
side of the uterine circulation. Conversely, failure
shown that exposure of normal villous explants to
of cytotrophoblasts to gain access to an adequate
hypoxia (1%O ) caused a 2-fold elevation in the
supply of maternal arterial blood may impair their
2
level of sFlt-1 secretion, as compared with 5% O
ability to differentiate into fully invasive cells. The
2
exposure. Nevertheless, when they pre-incubated
latter scenario could be a contributing factor to
human umbilical vein endothelial cells (HUVEC)
preeclampsia.
with either conditioned media from preeclampsia
The effects of hypoxia on the VEGF re-
placental explants or exogenous sFlt-1, cell migra-
Preeclampsia
ceptors were initially studied in endothelial cells.
in
tion was significantly reduced. The latter experi-
Gerber et al showed that hypoxia up-regulated the
ment suggests that sFlt-1 may also compromise
Flt-1 receptor expression in human umbilical vein
endovascular trophoblast (which converts into an
endothelial cells (HUVEC), whereas Flk-1/KDR
dvances
endothelial cell phenotype) migration and inva-
mRNA levels were unchanged or slightly re-
A
sion, thus causing defective spiral artery transfor-
pressed (68). They found an Flt-1 promoter region
al:
mation and in turn, hypoxia.
that included a sequence matching the hypoxia-in-
et
Earlier extensive studies were conduct-
ducible factor-1 (HIF) consensus binding site pre-
ed by Susan Fisher’s group. They suggested that
viously found in other hypoxia-inducible genes
Bdolah
oxygen tension could also regulate cytotropho-
such as the VEGF gene and erythropoietin gene.
blasts ability to differentiate and, as a consequen-
How should the algorithm of preec-
ce, express proteins that are critical for placental
lampsia pathophysiology be? Placental hypoxia ®
invasion (52). Preeclampsia, on the other hand, is
sFlt-1 rise ® maternal endothelial dysfunction ®
associated with failure of cytotrophoblasts to in-
preeclampsia? Or maybe: sFlt-1 rise ® Placental
vade the spiral arterioles (69). Therefore, they hy-
pothesized that in this disease the events that nor-
hypoxia ® maternal endothelial dysfunction ®
mally take place during the first trimester of preg-
preeclampsia? In other words, which is the chick-
nancy, which convert the maternal-fetal interface
en and which is the egg (26)?
from a relatively hypoxic environment to one that
What additional data supports hypoxia,
is relatively well oxygenated, fail to occur. In two
as the primary placental insult, causing pre-
different invasion assays, 2% oxygen-exposed
eclampsia? Susan Fisher’s group studied a primary
cytotrophoblasts showed markedly reduced inva-
culture of cytotrophoblasts isolated from placentas
sion, similar to in vivo cytotrophoblast behavior,
of 3rd trimester HELLP syndrome patients (72). As
as seen in preeclamptic placental sections. Hypo-
compared with controls, the sFlt-1 levels in the
xia was also shown to alter the normal pattern of
cytotrophoblast-conditioned medium released ap-
cytotrophoblast integrin switching. Cytotropho-
proximately twice the amount of the soluble re-
blasts maintained in 20% O rapidly up-regulated
2
ceptor. Based on in vitro studies, connecting hypo-
their expression of integrins a a marker first ex-
5,
xia to trophoblast changes characteristic of pre-
pressed just after these cells leave their basement
eclamptic placentas it is likely that the severe
membrane, and a , which is expressed later, as the
preeclamptic placentas were hypoxic, and there-
1
cells invade the uterus (70,71). Cells exposed to
fore sFlt-1 increased secretion is secondary to
2% O completed the initial stage of this process
hypoxia. The study of Nagamatsu et al (25) sup-
2
by expressing a , but failed to express a , suggest-
ports such a conclusion. A negative correlation be-
5
1
ing that they can initiate, but not complete, the
tween oxygen tension and sFlt-1 concentration in
normal integrin switching program.
the cytotrophoblast cell culture medium was ob-
served. These data are based on in vitro experi-
Fisher suggested that the effects of the
ments and, therefore, cannot be interpreted as the
relatively hypoxic environment on the prolifera-
actual in vivo mechanism responsible for pre-
tive capacity of cytotrophoblasts before 10 weeks
eclampsia initiation.
of gestation could account for the discrepancy be-
tween the rapid increase in placental mass and the
What additional evidence strengthens
slower growth of the embryo proper. Relatively
the notion that an elevated level of sFlt-1 secreted
high oxygen tension promotes cytotrophoblast dif-
by the placenta is the primary insult in preeclamp-
ferentiation and explains these cells’ extensive
sia, preceding hypoxia? Decreased serum levels of
invasiveness of the arterial rather than the venous
PlGF have been clearly shown to precede the clin-
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Croat Med J
2005;46(5):728-736
ical signs of preeclampsia (17). First trimester re-
rise ® maternal endothelial dysfunction ® pre-
duction of serum PlGF concentration in patients
eclampsia.
destined to develop preeclampsia (47) may sug-
gest that placental angiogenesis imbalance is the
Conclusions, Future Research and
Bdolah
primary cause of abnormal placentation, which
Treatment Options
eventually leads to preeclampsia in the second or
third trimester of pregnancy. Moreover, sFlt-1 has
The maternal syndrome of preeclampsia
et
al:
been shown in vitro to interfere with cytotropho-
is thought to be secondary to abnormal placen-
blast invasion and differentiation (72). Preliminary
tation and excess placental production of sFlt-1
A
dvances
experiments in our laboratory with a rat chorio-
(Fig. 1). It is still unclear whether placental hypo-
carcinoma cell line (Rcho-1) have demonstrated
xia or excess sFlt-1 production is the trigger event
that sFlt-1 may suppress the differentiation process
in the pathogenesis of preeclampsia, though most
in
of rat cytotrophoblast cells into giant trophoblast
current studies provide evidence that the massive
Preeclampsia
cells, the rodent equivalent of the human endo-
sFlt-1 production noted during clinical pre-
vascular invasive trophoblast (73). If these effects
eclampsia may be secondary to placental hypoxia.
prove genuine in other trophoblast assays as well,
Further studies in transgenic animals looking at lo-
we may be able to speculate that angiogenesis bal-
cal sFlt-1 over-expression in the placenta may
ance is crucial to the very first steps of differentia-
shed light on the role of sFlt-1 during early placen-
tion and invasion of cytotrophoblasts and maternal
tal development.
vessel remodeling. Hypoxia may, in turn, result in
Despite extensive research, preeclamp-
more sFlt-1 production, thus leading to a vicious
sia remains one of the leading causes of maternal
cycle of sFlt-1 production, eventually causing
mortality worldwide and yet, there is no reliable
preeclampsia (Fig. 2). Put differently, the pre-
screening test (74) or effective treatment to cure
eclampsia algorithm would look this way: sFlt-1
this disease. A prospective, multi-center trial to es-
rise ® angiogenesis imbalance ® failure of trop-
tablish the sensitivity, specificity, and predictive
hoblast invasion and physiological remodeling of
value of both serum sFlt-1/PlGF and urinary PlGF
uterine spiral arteries ® hypoxia ® further sFlt-1
as screening tests for preeclampsia in various pop-
ulations, including healthy women and in various
preeclampsia risk groups, may commence soon.
Genetic
Immunological
Other
Results of such a study will have a tremendous ef-
factors
factors
factors
fect on the delivery of optimal maternal and neo-
sFlt-1
natal care. Furthermore, the identification of circu-
PIGF
lating antiangiogenic factors such as sFlt-1 may fa-
Abnormal placentation
cilitate the development of new pharmacologic
VEGF
therapies that would be effective in treating or pre-
venting this devastating disease. That could make
Placental hypoxia
a gigantic step towards better feto-maternal well-
being.
sFlt-1, PIGF, VEGF
Acknowledgment
YB is supported by a fellowship from the Ameri-
can Physicians Fellowship for Medicine in Israel & The Ob-
Anti-angiogenic state, endothelial dysfunction
stetrics and Gynecology Foundation of the Department of
Ob/Gyn Beth Israel Deaconess Medical Center Harvard Medi-
cal School.
Preeclampsia
Figure 2. Hypothesis of the role of sFlt-1 and placental
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Benjamin P. Sachs
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Department of Obstetrics & Gynecology and
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Reproductive Sciences
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Beth Israel Deaconess Medical Center
way, in vivo. J Clin Invest. 1992;89:210-22.
Bdolah
330 Brookline Avenue-KS3182
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Boston, MA 02215, USA
ligands and receptors that regulate human cytotropho-
bsachs@bidmc.harvard.edu
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