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Hypertension Research

In Pregnancy

K. Kanasaki and M. Kanasaki


Angiogenic defects in preeclampsia:

What is known, and how are such
defects relevant to preeclampsia

Reprint request to:

Keizo Kanasaki, M.D., Ph.D.,
Diabetes & Endocrinology,
Kanazawa Medical University,
Uchinada, Ishikawa, 920-0293,
Key words:
2-ME2, angiogenesis, sFlt1,
soluble receptor, VEGF receptor
Received: March 31, 2013
Accepted: June 29, 2013

Keizo Kanasaki, Megumi Kanasaki

Department of Diabetology and Endocrinology, Kanazawa Medical University

Preeclampsia is a devastating pregnancy-associated hypertensive syndrome. Although it is quite common, the

pathophysiology of preeclampsia is not yet clear and remains a disease of theory. Angiogenic defect hypotheses have
been intensively investigated, and some biomarkers have been independently analyzed as pathogenic clinical target
molecules without direct proof of their roles in preeclampsia. In this review, we assessed an up-to-date list of proposed
angiogenic defects for their relevance to preeclampsia. In addition, we introduce our working hypothesis of
preeclampsia pathophysiology, which involves interactions between metabolic and angiogenic defects.

Preeclampsia is a devastating syndrome characterized
by pregnancy-specific new-onset hypertension and
proteinuria. The overall incidence is approximately
58% of all pregnant women.1) Presenting symptoms can
be highly variable, starting from seemingly innocuous
complaints, such as heartburn or headache, and
eventually progressing to multi-systemic, life-threatening
dysfunctions, such as HELLP (hemolysis, elevated liver
function tests, low platelets) syndrome and eclampsia
(characterized by seizures).1) Therapeutic options are
limited at present; only delivering the baby provides an
absolute cure for worsening maternal symptoms. The
preeclampsia-associated newborn is often premature,
resulting in significant perinatal morbidity and mortality,
as well as effects on future development. It is estimated
that approximately 15% of preterm births are due to
Despite its fairly high incidence,1) our understanding
of preeclampsia pathophysiology is limited. Preeclampsia
has been recognized as a disease of theories because
several hypotheses have been advanced to explain its
pathogenesis. Among these hypotheses, the angiogenic
molecular defects theory has received considerable
attention, with some pilot studies initiated to target these
defects as pathogenic molecules. Most of the angiogenic
defects theory is based on clinical molecular marker
observations using cDNA-carrying viral vectors to

assess overexpression of foreign proteins. In this review,

we analyze whether the angiogenic molecular defects
theory is pathophysiologically supported by enough
clinical/biological evidence to explain preeclampsia.
We also introduce a potential interaction between
catechol-metabolizing defects and angiogenic regulation
deficiency in preeclampsia pathogenesis.

Placental defects and placental hypoxia

It is widely accepted that placental hypoxia can
trigger preeclampsia by initiating a sequence of events
that ultimately results in the maternal symptoms of
hypertension and proteinuria.1) Placental hypoxia can
be caused by diverse factors, such as maternal hypoxia,
maternal hypotension, and placental ischemia, but in the
context of preeclampsia, it is considered secondary to
defective placental vasculature.2)
The placenta is an essential organ for exchanging
waste and nutrients between mother and fetus. An
embryonic cell known as the cytotrophoblast is
essential for proper development of the placenta and
its vasculature. One cytotrophoblast derivative, the
extravillous cytotrophoblast, invades the uterine matrix,
makes direct contact with the maternal blood supply, and
subsequently replaces the smooth muscle cell layer of the
maternal vasculature.1) This remodeling via trophoblast
invasion results in vessels that are maternal-derived
and vasoactive substance-resistant. Because the vessels

Hypertension Research in Pregnancy 2013 Japan Society for the Study of Hypertension in Pregnancy

Hypertens Res Pregnancy 2013; 1: 5765


Angiogenesis and preeclampsia

are independent from the influence of maternal blood
pressure regulation, they tend to be dilated with low
resistance.1) Preeclampsia is characterized by shallow
trophoblast invasion. As a result, placental circulation
does not provide sufficient blood supply to meet
embryonic demand due to high resistance, non-dilated
vessels that reflect inadequate remodeling of maternal
spiral arteries.1)
How does placental hypoxia cause the maternal
symptoms of preeclampsia? This important question is
still openly debated. One theory is that reduced placental
perfusion results in the production of numerous placentaderived circulatory agents, which cause the maternal

Discovery of angiogenic imbalance in

Much attention has been given to the topic of angiogenic
imbalance in preeclampsia pathogenesis. However,
this theory is rather complicated due to the biology of
vascular endothelial growth factor (VEGF) (Figure 1).
Angiogenic abnormalities in preeclampsia were
first reported by Selvaggi et al., who used the chick
embryo chorioallantoic membrane angiogenic assay
to demonstrate that decidua in preeclamptic women
exhibited higher angiogenic activity than in a normal
pregnancy.3,4) Although the angiogenesis imbalance
theory is popular, these important findings have not
been described well in the literature. Such an enhanced

angiogenesis signal could likely be a compensatory

reaction to shallow extravillous trophoblast invasion and
associated perfusion defects and placental hypoxia.3,4)
Selgaggis findings were important because they revealed
that hypoxic placental decidua in preeclampsia exhibited
enhanced angiogenic activity.
Despite this evidence of enhanced angiogenic activity
in the preeclamptic placenta, preeclampsia has been
considered an anti-angiogenic state,58) although this has
not been proven yet. This theoretical shift in preeclampsia
pathogenesis, from angiogenic to anti-angiogenic, is
linked to the implication of neutralizing soluble VEGF
receptors. Soluble VEGF type1 receptor (also known
as soluble Fms-like tyrosine kinase-1 [sFlt-1]) has
been hypothesized as an important candidate molecule
associated with preeclampsia pathogenesis.9 12) In 1998,
Clark et al. discovered that serum from pregnant women
contained a VEGF-binding protein not present in serum
from men or non-pregnant women.13) Vuorela et al.
initially reported that VEGF was bound to soluble proteins
in amniotic fluid and maternal serum,9) later discovering
in 2000 that sFlt-1 was significantly elevated in the
amniotic fluid of women with preeclampsia.10) This work
was the seminal report implicating sFlt-1 in preeclampsia.
Fisher et al. later demonstrated that cytotrophoblasts
from preeclamptic placentas exhibited higher levels of
sFlt-1 in vitro compared to control cytotrophoblasts from
normal placentas.12) In 2003, Sugimoto et al. discovered
that injecting sFlt-1 protein or anti-VEGF antibodies into
mice resulted in proteinuria, likely via neutralization of

Binding protein
VEGF biological
activity (+)



VEGF biological
activity (-)
VEGF biological
activity (high? or low?)
Other unknown
binding partners

Figure 1. VEGFbindingpartnersandtheireffectsonVEGFactivity.
Vascular endothelial growth factor (VEGF) can bind to several molecules, and certain interactions do not detectably
alter VEGF activity, as assessed by a commercially available ELISA assay.

Hypertens Res Pregnancy 2013; 1: 5765

K. Kanasaki and M. Kanasaki

the bioavailable form of VEGF.14) Later in the same year,
Maynard et al. reported that the concentration of sFlt-1
in serum from preeclamptic women was higher compared
to women with normal pregnancies and that viral
vector-mediated delivery of human sFlt-1 cDNA caused
endotheliosis and hypertension in male, non-pregnant
female and pregnant female rats.15) Their study also
demonstrated that serum from preeclamptic women was
anti-angiogenic compared to normal control pregnancies
when analyzed by the endothelial tube-formation assay.
Notably, incubation with both 10 ng/ml VEGF (a
concentration approximately 1,000 higher than normal
pregnancy) and 10 ng/ml placental growth factor (PlGF)
(a concentration approximately 25 higher than normal
pregnancy) abolished this anti-angiogenic property of
preeclamptic serum.15)

Mechanistic theory of angiogenic

balance-induced preeclampsia
As described above, angiogenic and anti-angiogenic
imbalance theories have been intensively investigated.
However, the important fundamental question that
remains is whether VEGF biological activity is high or
low in preeclampsia.
The study described above by Maynard et al., showing
that adenoviral-mediated sFlt-1 overexpression resulted
in preeclampsia-like symptoms in rats,15) led to the
widespread but untested concept that sFlt-1 may play
a causal role in preeclampsia pathogenesis. Maynards
theory is mainly based on the anti-angiogenic hypothesis
of preeclampsia; however, many fundamental issues need
to be addressed in this model. First, the concentration
achieved by adenoviral-mediated overexpression of
human sFlt-1 is approximately 215.5 ng/ml in rats,
which is significantly higher compared to that observed
in severe preeclampsia (7.6 ng/ml).15) The authors
also described a lower concentration of human sFlt-1
(7.3 3.2 ng/ml, also produced by adenoviral vector)
that caused a milder preeclampsia phenotype in rats,
although it is not clear whether these experiments were
performed in pregnant, male, or female rats.15) The
second question is the relevance of sFlt-1 species and
isoforms used in these studies. It is unknown which
sFlt-1 isoforms increase with preeclampsia, a highly
important point when producing appropriate animal
models. For example, the primate-specific variant sFlt1-14 has been reported in preeclampsia16) but not tested
as a preeclampsia candidate molecule in animal models.
Sugimoto et al. employed a human sFlt-1/human IgGFc
chimera that was intravenously injected into mice.14)
Maynards work and subsequent adenovirus-based
preeclampsia rat models used mouse sFlt-1 (lacking 31
amino acid carboxyl residues) and immunoglobulin-like

loop 4,15) which is essential for receptor dimerization of

the extracellular domains of sFlt-1.17,18) To solve these
problems, Suzuki et al. used an adenovirus to express
full-length mouse sFlt-1 in a mouse model.19) They
found that only a very high concentration of adenovirus
vector (2 109, compared to 1 108 in Maynards work)
produced a preeclampsia-like phenotype with 100 ng/ml
of plasma sFlt-1 in mice; however, a concentration of
1 109 could induce a similar concentration of plasma
sFlt-1 in mice and cause gestational proteinuria without
gestational hypertension.19) Moreover, the hypertension
observed in this model was attenuated by mouse VEGF
or PlGF adenovirus administration, but proteinuria
was not.19) Adenovirus infection results in serious
liver damage,20,21) which could not be ignored as a
confounding factor in all adenoviral preeclampsia models.
In this regard, Kumasawa et al. established an elegant
preeclampsia mouse model by transducing a lentiviral
vector expressing human sFlt-1 into zona pellucida-free
blastocysts to establish pregnancy placenta-specific sFlt-1
expression in mice.22) By overexpressing human sFlt-1,
their model somewhat mimicked pregnancy induced
hypertension with proteinuria.22) Hypertension was
rescued by administration of the cholesterol-lowering
agent pravastatin, likely associated with elevation of
PlGF levels.22) Pravastatin showed a trend of attenuated
proteinuria in their model.22)
The assumption was that so-called free VEGF, which
can be measured with enzyme-linked immunosorbent
assay (ELISA), is the only bioactive form of VEGF;
however, this has never been biologically proven,
and recent evidence has challenged this view.23) High
VEGF levels have been reported for preeclampsia
in several biological fluids, including plasma, serum
and amniotic fluid, by radioimmunoassay (RIA) or
capture enzyme immunoassay.23) However, commercially
available VEGF ELISA kits revealed suppressed VEGF
levels in preeclampsia compared to normal pregnancy.23)
This discrepancy in measurements with different
methodologies led researchers to speculate that VEGFbinding molecules interfere with antibody binding in
ELISAs, which may measure unbound free VEGF
levels.15,23) Whether free VEGF is the only active form
of VEGF has not been experimentally proven. When
estimated using RIA, the VEGF concentration was more
than 20 higher than that of sFlt-1, even in preeclampsia;
when estimated using ELISA, VEGF levels were much
lower than those of sFlt-1, even in normal pregnancy.23)
Therefore, although it is true that sFlt-1 levels are higher
in most cases of preeclampsia,5,15,24) sFlt-1 levels cannot
completely explain the reduced VEGF levels, as observed
by ELISA; rather, it must be due to other molecules
that bind VEGF without affecting its biological activity.
Some reports have indicated that VEGF activity is high
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Angiogenesis and preeclampsia

in preeclampsia.2527) One molecule that may bind
VEGF is 2-macroglobulin, and this interaction does
not influence VEGF-induced receptor activation.28,29)
Moreover, alternative splice variants, such as VEGF165b,
act as weak agonists for the angiogenic receptor
VEGF receptor 2. VEGF165b inhibits VEGF165-induced
intracellular signaling and thus plays an antagonistic role
in VEGF165-mediated angiogenesis.23,25,30) Nevertheless,
the biological activity of VEGF is often confused with
the unbound form of VEGF in the literature. This point
needs to be carefully investigated in future research
(Figure 1).

Is there direct evidence of angiogenic

imbalance causing human preeclampsia?
Pregnant women who develop preeclampsia often
exhibit high sFlt-1 levels with low free VEGF (but not
total VEGF), as measured by ELISA, and low PlGF.
Drug administration in pregnant women is challenging
due to potential teratogenicity and toxicity. Therefore,
the ideal preeclampsia therapy would remove some
pathogenic factors from circulatory blood without
administering a drug. Thadhani et al. developed a
unique way to test the anti-angiogenic theory of
preeclampsia.31) sFlt-1 is a positively charged circulatory
protein, and they focused on this characteristic as a
therapeutic target.31) Based on the unproven sFlt-1
theory, they tried to eliminate positively-charged sFlt-1
from preeclamptic serum using an extracorporeal affinity
adsorption column,31) specifically, clinically-available
negatively-charged dextran sulfate columns (DSCs)
which would theoretically absorb positively-charged
sFlt-1. In their analysis, DSCs decreased sFlt-1 levels in
the plasma of preeclamptic women from 13,471 3,358
pg / ml to 10,825 2,918 pg / ml (n = 5, 20.1 5.1%
reduction; mean s.d.).31) This decrease in sFlt-1 was
associated with a 56.8 6.7% reduction in the urine
protein-creatinine ratio (3,326 2,306 to 1,349 811).31)
Although an interesting idea, this pilot study did not
provide information about how the system works.
sFlt-1 is not the only positively-charged molecule with
a potential pathogenic role in preeclampsia. Fibrinogen
is also positively-charged and highly relevant to
blood rheology and coagulation, and DSC apheresis
decreases its content by 18.0 11.3%.31) Low density
lipoprotein cholesterol, other inflammatory proteins,
and immunoglobulins (especially angiotensin receptor
autoantibodies) could also be removed from preeclamptic
maternal serum by DSCs. Performing apheresis itself may
alter blood pressure and rheology. Wang et al. elegantly
demonstrated that the pregnancies of 9 very preterm
preeclamptic women were prolonged 3 to 49 additional
days by utilizing apheresis with heparin-mediated

Hypertens Res Pregnancy 2013; 1: 5765

extracorporeal low-density lipoprotein precipitation

(H.E.L.P.) instead of DSCs.32) H.E.L.P. apheresis in
preeclamptic women was associated with reductions in
tumor necrosis factor-, soluble vascular cell adhesion
molecule 1, E-selectin, endothelin 1, lipopolysaccharidebinding protein, homocysteine, fibrinogen and C-reactive
protein.32) H.E.L.P. also reduced apolipoprotein B
containing lipoproteins by almost 50% in preeclamptic
women.32) H.E.L.P. apheresis is likely not associated
with the reduction in sFlt-1 because, as Thadhani et al.
stated, it is less effective than DSCs in reducing sFlt1.31) DSC apheresis reduces lipoprotein levels; therefore,
interpreting the DSC data described above is more
complicated than simple sFlt-1 removal.

COMT/2-ME defects and preeclampsia:

not as simple as the angiogenic
imbalance theory
Catechol-O-methyltransferase (Comt) is a candidate gene
in various psychiatric disorders, such as schizophrenia,
that encodes a catabolic enzyme fundamental in the
metabolism of diverse catechols, including catecholamines
and catecholestrogens. Estradiol is metabolized by
cytochrome p450, and synthesized 17-hydroxyestradiol
(a catecholestrogen) is a substrate for COMT. This
catecholestradiol is then metabolized by COMT into
2-methoxyestradiol (2-ME), in a rate-limiting step. 2-ME
inhibits hypoxia-inducible factor-1 (HIF-1), possibly
by destabilizing microtubules,33) allowing it to act as an
anti-angiogenic molecule in a context-dependent manner.
Clinical evaluations are currently underway to investigate
2-ME as a new oral therapeutic agent in the treatment of
cancer.34 36) Normal pregnant women exhibit increased
concentrations of 2-ME that peak at term (from 215 nM
in the 1116th week of pregnancy to 1896.21 nM in the
3740th week of pregnancy).37)
Barnea et al. first reported the suppression of
placental COMT activity in preeclampsia in 1988,38)
but its significance was not mechanistically analyzed.
We recently demonstrated that deficiencies in COMT
and 2-ME are associated with placental hypoxia
and preeclampsia-like symptoms in mice.33) Comtdeficient mice (Comt- / -) display pregnancy induced
hypertension with proteinuria due to the absence of
2-ME, and administration of exogenous 2-ME (10 ng
subcutaneous injection from day 10 of pregnancy)
ameliorates hypertension, proteinuria, placental defects,
acute atherosis and glomerular and placental endothelial
damage in pregnant Comt- / - mice (Figure 2). The
preeclampsia-like phenotypes of pregnant Comt- / - mice
were largely corroborated by Stanley et al.,39) and any
differences could be explained by the mouse strain used
(SV129 in our study, C57BL/6 in the study by Stanley

K. Kanasaki and M. Kanasaki

Low placental
estradiol production

Placental hypoxia
Vascular defects
Angiogenic defects

Placental defects

Figure 2. WorkinghypothesisofCOMTdeficiencyandthepathogenesisofpreeclampsia.
Deficiency in catechol-O-methyltransferase (COMT) and the associated suppression of 2-methoxyestradiol (2-ME) levels
may result in hypoxia, vascular defects, hypoxia-inducible factor-1 alpha accumulation and inflammation in placentas.
These defects in placental homeostasis induce defects in both angiogenesis and the placenta. Placental defects lead to
suppression of estradiol production from placentas, resulting in further suppression of 2-ME. This vicious cycle leads
to preeclampsia.
et al.).33)
The underlying mechanisms of COMT and 2-ME
deficiency in the pathogenesis of a preeclampsia-like
phenotype may be linked to HIF-1 accumulation in
Comt-/- mice placentas (Figure 2), which is associated
with an enhanced inflammatory response and endothelial
damage. Suppression of hypoxia-induced HIF-1
accumulation by 2-ME is correlated with sFlt-1
suppression. 2-ME decreases NK cell accumulation and
interferon- production, ameliorating endothelial damage
in Comt-/- mice (Figure 2). 2-ME also induces trophoblast
invasion under hypoxic conditions by suppressing
HIF-1 and transforming growth factor-beta 3, likely
via trophoblast-to-endothelial-like cell conversion,
suggesting that 2-ME is a fundamental player in
maintaining placental homeostasis40) and supporting the
theory proposed by Caniggia et al.41) 2-ME may directly
function as a vasodilator and inhibit vasospasm42 45) in
pregnant women.
COMT enzymatic activity exhibits a trimodal frequency
distribution in human populations, which could be
explained by the presence of a functional polymorphism
in the coding sequence.46) One such polymorphism,
rs4680 (G-A nucleotide substitution), results in a
missense mutation of Val to Met at position COMT158.46)
Human COMTMet158 has a lower stability with a lower
enzymatic activity at physiological temperatures,46)
and approximately 2530% of humans possess this

polymorphism. Sata et al. reported that this functional

Comt polymorphism is associated with fetal growth
restriction.47) In addition to COMT,Met158 Nackley et al.
reported that haplotypes in multiple single nucleotide
polymorphisms (SNPs) of the Comt gene affect
COMT mRNA stability,48) suggesting that preeclampsia
susceptibility is associated with genomic alterations. It
must be emphasized that these hypotheses have yet to be
investigated, and other SNP analyses should be conducted
in multiple families in different population cohorts.
COMT deficiency cannot explain all phenotypical
preeclampsia variants in humans. Preeclampsia is likely
a heterogeneous collection of diverse pathomechanisms,
and COMT deficiency may only play a role in select
patient populations. For example, maternal COMTMet158
has been associated with preeclampsia in Korean and
Chinese cohorts,49,50) and a Norwegian population cohort
(HUNT2) study revealed that the maternal haplotype
for low COMT activity was associated with recurrent
preeclampsia.51) COMT activity may be driven by factors
other than SNPs, such as translational control or other
diverse pathways that lead to decreased activity.52,53)
Elevated homocysteine levels may be associated
with an increased risk of preeclampsia.54,55) The
mechanisms by which homocysteine contributes to
preeclampsia pathogenesis could be explained by
thrombosis of placental vasculature, inhibition of nitric
oxide production and general oxidative stress.56 59)
Hypertens Res Pregnancy 2013; 1: 5765


Angiogenesis and preeclampsia


B12, folate













Figure 3. Proposed hypothetical models in the interaction of hyperhomocysteinemia and preeclampsia onset via
Due to deficiencies in nutrition and activities of diverse enzymes, homocysteine levels are elevated. Homocysteine is
converted into s-adenosyl-homocysteine, an endogenous inhibitor of the catechol-O-methyltransferase (COMT) enzyme.
Therefore, women (or a combination of the placenta and pregnant mother) with SNPs associated with low COMT
enzymatic activity can exhibit severely suppressed COMT activity, leading to preeclampsia.
At normal concentrations, homocysteine primarily
proceeds along pathways associated with less reactive
oxidative methionine or cysteine; however, excess
homocysteine is increasingly converted to S-adenosyl
homocysteine (SAH), which is a potent endogenous
COMT inhibitor (Figure 3).60) If SAH accumulates
in sufficient concentrations to inhibit COMT activity,
O-methylation and inactivation of circulatory endogenous
catecholamines and other catechols decrease.60) An
increase in circulatory catecholamines can result in
sympathetic nervous system overactivation, leading
to an increased risk of cardiovascular morbidity and
Men with high serum homocysteine levels who
are also homozygous for the low-activity Comt gene
(Val158Met isoform; COMTLL) are at increased risk of
coronary heart disease.61) This finding may be relevant
to the pathogenesis of preeclampsia in women who
are homozygous for COMTLL. The possibility that
homocysteine-induced preeclampsia is dependent on
Comt genotype could be why hyperhomocysteinemia did
not lead to preeclampsia in previous animal studies with
wild-type rodents.62,63) It is also possible that placental,
but not maternal, or both Comt genotypes are much more
relevant to interactions with hyperhomocysteinemia.
hyperhomocysteinemia and low COMT activity provides

Hypertens Res Pregnancy 2013; 1: 5765

several potential pathomechanisms to explain why ComtLL

women or women with other haplotypes associated with
the low-activity COMT enzyme might be susceptible
to preeclampsia. Increased levels of homocysteine (and
SAH) could suppress COMT enzymatic activity and
subsequently reduce 2-ME production (Figure 3). Similar
to the findings in ComtLL men, hyperhomocysteinemia
may also contribute to the defective metabolism of
endogenous catecholamine levels in ComtLL women
or women with the other haplotypes. Furthermore,
deficiencies in potent homocysteine suppressors, such
as B vitamins and folic acid, could increase the risk
of preeclampsia.64 67) Folic acid supplementation in
the second trimester of pregnancy has been shown to
significantly reduce both serum homocysteine levels and
the risk of preeclampsia.68)
More research is required to clarify these results, as the
effects of folic acid therapy observed in this study could
have been related to the Comt phenotype. These results
could also pave the way to future studies that investigate
the interaction of Comt SNPs and other genetic defects
in the folic acid metabolism pathway, such as the
methylenetetrahydrofolate reductase (Mthfr) C677T
mutation,69) with the development of preeclampsia. In
this regard, both maternal and fetal Comt haplotypes
may be associated with a fetal Mthfr C677T mutation in
preeclampsia pathogenesis.70)

K. Kanasaki and M. Kanasaki

of preeclampsia
Placentas might exhibit an anti-angiogenic state with
compensatory induction of angiogenic signaling; however,
it is not clear whether systemically preeclamptic women
develop this state. Most evidence supporting the antiangiogenesis state hypothesis is based on biomarker
analysis rather than a mechanistically proven theory.58)
Because virus-mediated overexpression models may lead
us to overestimate preeclampsia pathogenesis,15,19,22,71 74)
it would be extremely useful to have a relevant genetic
mouse model. Such models may be phenotypically weak
compared to those generated by adenovirus transfer33);
however, weak but relevant phenotypes are common
in genetic disease models. Despite similarities in
general physiology, mouse disease models are not exact
phenocopies of human pathophysiology, and achieving a
complete copy is impossible. This limitation has been
well documented in genetic cancer models, in which
phenotypes between mice and men overlap, but the tumor
progression rate and associated outcomes can differ.
We must understand how to design proper, useful
animal models, and we should not make misinterpretations
based on the limitations of disease models. It is important
to understand the limitation of each animal model, even if
stronger phenotypes can be obtained via physiologically
irrelevant interventions. Angiogenic abnormalities and
the anti-angiogenic state in preeclampsia remain
unproven hypotheses. The differences between
molecular biomarker analyses must therefore be carefully
interpreted to understand the role of such biomarkers in
disease pathogenesis.
This review focused on the angiogenic abnormality
theory. Other highly relevant theories such as
inflammation75,76) and angiotensin receptor agonistic
autoantibodies 77) have also been suggested as
pathomechanisms of preeclampsia. Additionally, none
of the animal preeclampsia models exhibit metabolic
defects such as insulin resistance, which is an important
associated condition in human preeclampsia. Future
research is needed to identify mechanisms to understand
preeclampsia pathogenesis and establish relevant animal

The authors declare no conflict of interest in this work.
The authors laboratories are currently supported by
grants from the Japan Society for the Promotion of
Science to M.K. (24790329) and K.K. (23790381).
K.K. is also supported by a Grant for Promoted
Research (S2011-1, S2012-5) from Kanazawa Medical
University and several foundation grants, including the

Japan Research Foundation for Clinical Pharmacology,

the Daiichi-Sankyo Foundation of Life Science, the
Ono Medical Research Foundation, the NOVARTIS
Foundation (Japan) for the Promotion of Science, the
Takeda Science Foundation and the Banyu Life Science

1. Kanasaki K, Kalluri R. The biology of preeclampsia. Kidney Int.
2009; 76: 831 837.
2. Sibai B, Dekker G, Kupferminc M. Pre-eclampsia. Lancet. 2005;
365: 785 799.
3. Ribatti D, Loverro G, Vacca A, Greco P, Roncali L, Selvaggi L.
Expression of tenascin is related to angiogenesis in pre-eclampsia.
Eur J Clin Invest. 1998; 28: 373 378.
4. Selvaggi L, Ribatti D, Loverro G, Sorino S, Greco P, Roncali L.
Angiogenesis in pre-eclampsia. Eur J Obstet Gynecol Reprod Biol.
1995; 59: 83 89.
5. Levine RJ, Maynard SE, Qian C, et al. Circulating angiogenic
factors and the risk of preeclampsia. N Engl J Med. 2004; 350:
672 683.
6. Levine RJ, Thadhani R, Qian C, et al. Urinary placental growth
factor and risk of preeclampsia. JAMA. 2005; 293: 77 85.
7. Maynard SE, Karumanchi SA. Angiogenic factors and preeclampsia.
Semin Nephrol. 2011; 31: 33 46.
8. Powe CE, Levine RJ, Karumanchi SA. Preeclampsia, a disease of
the maternal endothelium: the role of antiangiogenic factors and
implications for later cardiovascular disease. Circulation. 2011;
123: 2856 2869.
9. Vuorela-Vepsalainen P, Alfthan H, Orpana A, Alitalo K, Stenman
UH, Halmesmaki E. Vascular endothelial growth factor is bound
in amniotic fluid and maternal serum. Hum Reprod. 1999; 14:
1346 1351.
10. Vuorela P, Helske S, Hornig C, Alitalo K, Weich H, Halmesmaki
E. Amniotic fluid--soluble vascular endothelial growth factor
receptor-1 in preeclampsia. Obstet Gynecol. 2000; 95: 353357.
11. Helske S, Vuorela P, Carpen O, Hornig C, Weich H, Halmesmaki
E. Expression of vascular endothelial growth factor receptors 1, 2
and 3 in placentas from normal and complicated pregnancies. Mol
Hum Reprod. 2001; 7: 205 210.
12. Zhou Y, McMaster M, Woo K, et al. Vascular endothelial growth
factor ligands and receptors that regulate human cytotrophoblast
survival are dysregulated in severe preeclampsia and hemolysis,
elevated liver enzymes, and low platelets syndrome. Am J Pathol.
2002; 160: 1405 1423.
13. Clark DE, Smith SK, He Y, et al. A vascular endothelial growth
factor antagonist is produced by the human placenta and released
into the maternal circulation. Biol Reprod. 1998; 59: 1540 1548.
14. Sugimoto H, Hamano Y, Charytan D, et al. Neutralization of
circulating vascular endothelial growth factor (VEGF) by antiVEGF antibodies and soluble VEGF receptor 1 (sFlt-1) induces
proteinuria. J Biol Chem. 2003; 278: 12605 12608.
15. Maynard SE, Min JY, Merchan J, et al. Excess placental soluble
fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial
dysfunction, hypertension, and proteinuria in preeclampsia. J Clin
Invest. 2003; 111: 649 658.
16. Sela S, Itin A, Natanson-Yaron S, et al. A novel human-specific

Hypertens Res Pregnancy 2013; 1: 5765


Angiogenesis and preeclampsia

















soluble vascular endothelial growth factor receptor 1: cell-typespecific splicing and implications to vascular endothelial growth
factor homeostasis and preeclampsia. Circ Res. 2008; 102: 1566
Barleon B, Totzke F, Herzog C, et al. Mapping of the sites for
ligand binding and receptor dimerization at the extracellular
domain of the vascular endothelial growth factor receptor FLT-1. J
Biol Chem. 1997; 272: 10382 10388.
Shinkai A, Ito M, Anazawa H, Yamaguchi S, Shitara K, Shibuya
M. Mapping of the sites involved in ligand association and
dissociation at the extracellular domain of the kinase insert
domain-containing receptor for vascular endothelial growth factor.
J Biol Chem. 1998; 273: 31283 31288.
Suzuki H, Ohkuchi A, Matsubara S, et al. Effect of recombinant
placental growth factor 2 on hypertension induced by full-length
mouse soluble fms-like tyrosine kinase 1 adenoviral vector in
pregnant mice. Hypertension. 2009; 54: 1129 1135.
Hoff CM, Margetts PJ. Adenovirus-based transient expression
systems for peritoneal membrane research. Perit Dial Int. 2006; 26:
Mahasreshti PJ, Kataram M, Wang MH, et al. Intravenous delivery
of adenovirus-mediated soluble FLT-1 results in liver toxicity. Clin
Cancer Res. 2003; 9: 2701 2710.
Kumasawa K, Ikawa M, Kidoya H, et al. Pravastatin induces
placental growth factor (PGF) and ameliorates preeclampsia in a
mouse model. Proc Natl Acad Sci U S A. 2011; 108: 1451 1455.
Bates DO. An unexpected tail of VEGF and PlGF in pre-eclampsia.
Biochem Soc Trans. 2011; 39: 1576 1582.
Levine RJ, Lam C, Qian C, et al. Soluble endoglin and other
circulating antiangiogenic factors in preeclampsia. N Engl J Med.
2006; 355: 9921005.
Bills VL, Salmon AH, Harper SJ, et al. Impaired vascular
permeability regulation caused by the VEGF(1)(6)(5)b splice
variant in pre-eclampsia. BJOG. 2011; 118: 1253 1261.
Brockelsby J, Hayman R, Ahmed A, Warren A, Johnson I, Baker
P. VEGF via VEGF receptor-1 (Flt-1) mimics preeclamptic
plasma in inhibiting uterine blood vessel relaxation in pregnancy:
implications in the pathogenesis of preeclampsia. Lab Invest. 1999;
79: 11011111.
Brockelsby JC, Anthony FW, Johnson IR, Baker PN. The effects of
vascular endothelial growth factor on endothelial cells: a potential
role in preeclampsia. Am J Obstet Gynecol. 2000; 182: 176 183.
Soker S, Svahn CM, Neufeld G. Vascular endothelial growth factor
is inactivated by binding to alpha 2-macroglobulin and the binding
is inhibited by heparin. J Biol Chem. 1993; 268: 7685 7691.
Bhattacharjee G, Asplin IR, Wu SM, Gawdi G, Pizzo SV. The
conformation-dependent interaction of alpha 2-macroglobulin with
vascular endothelial growth factor. A novel mechanism of alpha
2-macroglobulin/growth factor binding. J Biol Chem. 2000; 275:
Bills VL, Varet J, Millar A, Harper SJ, Soothill PW, Bates DO.
Failure to up-regulate VEGF165b in maternal plasma is a first
trimester predictive marker for pre-eclampsia. Clin Sci (Lond).
2009; 116: 265272.
Thadhani R, Kisner T, Hagmann H, et al. Pilot study of
extracorporeal removal of soluble fms-like tyrosine kinase 1 in
preeclampsia. Circulation. 2011; 124: 940 950.
Wang Y, Walli AK, Schulze A, et al. Heparin-mediated
extracorporeal low density lipoprotein precipitation as a possible

Hypertens Res Pregnancy 2013; 1: 5765

















therapeutic approach in preeclampsia. Transfus Apher Sci. 2006;

35: 103 110.
Kanasaki K, Palmsten K, Sugimoto H, et al. Deficiency in catecholO-methyltransferase and 2-methoxyoestradiol is associated with
pre-eclampsia. Nature. 2008; 453: 1117 1121.
Bruce JY, Eickhoff J, Pili R, et al. A phase II study of
2-methoxyestradiol nanocrystal colloidal dispersion alone and in
combination with sunitinib malate in patients with metastatic renal
cell carcinoma progressing on sunitinib malate. Invest New Drugs.
2012; 30: 794 802.
Kulke MH, Chan JA, Meyerhardt JA, et al. A prospective phase
II study of 2-methoxyestradiol administered in combination with
bevacizumab in patients with metastatic carcinoid tumors. Cancer
Chemother Pharmacol. 2011; 68: 293 300.
Harrison MR, Hahn NM, Pili R, et al. A phase II study of
2-methoxyestradiol (2ME2) NanoCrystal(R) dispersion (NCD)
in patients with taxane-refractory, metastatic castrate-resistant
prostate cancer (CRPC). Invest New Drugs. 2011; 29: 14651474.
Berg D, Sonsalla R, Kuss E. Concentrations of 2-methoxyoestrogens
in human serum measured by a heterologous immunoassay with
an 125I-labelled ligand. Acta Endocrinol (Copenh). 1983; 103:
282 288.
Barnea ER, MacLusky NJ, DeCherney AH, Naftolin F. Catecholo-methyl transferase activity in the human term placenta. Am J
Perinatol. 1988; 5: 121 127.
Stanley JL, Andersson IJ, Poudel R, et al. Sildenafil citrate rescues
fetal growth in the catechol-O-methyl transferase knockout mouse
model. Hypertension. 2012; 59: 1021 1028.
Lee SB, Wong AP, Kanasaki K, et al. Preeclampsia:
2-methoxyestradiol induces cytotrophoblast invasion and vascular
development specifically under hypoxic conditions. Am J Pathol.
2010; 176: 710 720.
Caniggia I, Mostachfi H, Winter J, et al. Hypoxia-inducible
factor-1 mediates the biological effects of oxygen on human
trophoblast differentiation through TGFbeta(3). J Clin Invest.
2000; 105: 577 587.
Hill BJ, Gebre S, Schlicker B, Jordan R, Necessary S.
Nongenomic inhibition of coronary constriction by 17ss-estradiol,
2-hydroxyestradiol, and 2-methoxyestradiol. Can J Physiol
Pharmacol. 2010; 88: 147 152.
Gui Y, Zheng XL, Zheng J, Walsh MP. Inhibition of rat aortic
smooth muscle contraction by 2-methoxyestradiol. Am J Physiol
Heart Circ Physiol. 2008; 295: H1935 1942.
Yan J, Chen C, Lei J, et al. 2-methoxyestradiol reduces
cerebral vasospasm after 48 hours of experimental subarachnoid
hemorrhage in rats. Exp Neurol. 2006; 202: 348 356.
Tofovic SP, Salah EM, Mady HH, Jackson EK, Melhem MF.
Estradiol metabolites attenuate monocrotaline-induced pulmonary
hypertension in rats. J Cardiovasc Pharmacol. 2005; 46: 430437.
Tunbridge EM, Harrison PJ, Weinberger DR. Catechol-omethyltransferase, cognition, and psychosis: Val158Met and
beyond. Biol Psychiatry. 2006; 60: 141 151.
Sata F, Yamada H, Suzuki K, et al. Functional maternal catecholO-methyltransferase polymorphism and fetal growth restriction.
Pharmacogenet Genomics. 2006; 16: 775 781.
Nackley AG, Shabalina SA, Tchivileva IE, et al. Human catecholO-methyltransferase haplotypes modulate protein expression by
altering mRNA secondary structure. Science. 2006; 314: 1930

K. Kanasaki and M. Kanasaki

49. Lim JH, Kim SY, Kim do J, et al. Genetic polymorphism of
catechol-O-methyltransferase and cytochrome P450c17alpha in
preeclampsia. Pharmacogenet Genomics. 2010; 20: 605 610.
50. Liang S, Liu X, Fan P, et al. Association between Val158Met
functional polymorphism in the COMT gene and risk of
preeclampsia in a Chinese population. Arch Med Res. 2012; 43:
51. Roten LT, Fenstad MH, Forsmo S, et al. A low COMT activity
haplotype is associated with recurrent preeclampsia in a Norwegian
population cohort (HUNT2). Mol Hum Reprod. 2011; 17: 439
52. Lott SA, Burghardt PR, Burghardt KJ, Bly MJ, Grove TB,
Ellingrod VL. The influence of metabolic syndrome, physical
activity and genotype on catechol-O-methyl transferase promoterregion methylation in schizophrenia. Pharmacogenomics J. 2013:
13: 264271.
53. Schreiner F, El-Maarri O, Gohlke B, et al. Association of COMT
genotypes with S-COMT promoter methylation in growthdiscordant monozygotic twins and healthy adults. BMC Med
Genet. 2011; 12: 115.
54. Baksu A, Taskin M, Goker N, Baksu B, Uluocak A. Plasma
homocysteine in late pregnancies complicated with preeclampsia
and in newborns. Am J Perinatol. 2006; 23: 31 35.
55. Maruotti G, Del Bianco A, Amato AN, Lombardi L, Fulgeri
AM, Pietropaolo F. [Preeclampsia and high serum levels of
homocysteine]. Minerva Ginecol. 2005; 57: 165 170.
56. Mao D, Che J, Li K, et al. Association of homocysteine,
asymmetric dimethylarginine, and nitric oxide with preeclampsia.
Arch Gynecol Obstet. 2010; 282: 371 375.
57. Hubel CA. Oxidative stress in the pathogenesis of preeclampsia.
Proc Soc Exp Biol Med. 1999; 222: 222 235.
58. Roberts JM, Hubel CA. Is oxidative stress the link in the two-stage
model of pre-eclampsia? Lancet. 1999; 354: 788 789.
59. Middeldorp S. Thrombophilia and pregnancy complications: cause
or association? J Thromb Haemost. 2007; 5 (suppl 1): 276 282.
60. Zhu BT. Catechol-O-Methyltransferase (COMT)-mediated
methylation metabolism of endogenous bioactive catechols
and modulation by endobiotics and xenobiotics: importance in
pathophysiology and pathogenesis. Curr Drug Metab. 2002; 3:
61. Voutilainen S, Tuomainen TP, Korhonen M, et al. Functional
COMT Val158Met polymorphism, risk of acute coronary events
and serum homocysteine: the kuopio ischaemic heart disease risk
factor study. PLoS One. 2007; 2: e181.
62. Falcao S, Bisotto S, Gutkowska J, Lavoie JL. Hyperhomocysteinemia
is not sufficient to cause preeclampsia in an animal model: the
importance of folate intake. Am J Obstet Gynecol. 2009; 200: 198.
63. Kassab SE, Abu-Hijleh MF, Al-Shaikh HB, Nagalla DS.
Hyperhomocysteinemia in pregnant rats: effects on arterial













pressure, kidneys and fetal growth. Eur J Obstet Gynecol Reprod

Biol. 2005; 122: 177 181.
Braekke K, Ueland PM, Harsem NK, Karlsen A, Blomhoff R, Staff
AC. Homocysteine, cysteine, and related metabolites in maternal
and fetal plasma in preeclampsia. Pediatr Res. 2007; 62: 319324.
Guven MA, Coskun A, Ertas IE, Aral M, Zencirci B, Oksuz
H. Association of maternal serum CRP, IL-6, TNF-alpha,
homocysteine, folic acid and vitamin B12 levels with the severity
of preeclampsia and fetal birth weight. Hypertens Pregnancy. 2009;
28: 190 200.
Sanchez SE, Zhang C, Rene Malinow M, Ware-Jauregui S,
Larrabure G, Williams MA. Plasma folate, vitamin B(12), and
homocyst(e)ine concentrations in preeclamptic and normotensive
Peruvian women. Am J Epidemiol. 2001; 153: 474 480.
Herrmann W, Isber S, Obeid R, Herrmann M, Jouma M.
Concentrations of homocysteine, related metabolites and
asymmetric dimethylarginine in preeclamptic women with poor
nutritional status. Clin Chem Lab Med. 2005; 43: 1139 1146.
Wen SW, Chen XK, Rodger M, et al. Folic acid supplementation
in early second trimester and the risk of preeclampsia. Am J Obstet
Gynecol. 2008; 198: 45.e1 7.
Ray JG, Laskin CA. Folic acid and homocyst(e)ine metabolic
defects and the risk of placental abruption, pre-eclampsia and
spontaneous pregnancy loss: A systematic review. Placenta. 1999;
20: 519 529.
Hill LD, York TP, Kusanovic JP, et al. Epistasis between
COMT and MTHFR in Maternal-Fetal Dyads Increases Risk for
Preeclampsia. PLoS One. 2011; 6: e16681.
Venkatesha S, Toporsian M, Lam C, et al. Soluble endoglin
contributes to the pathogenesis of preeclampsia. Nat Med. 2006;
12: 642 649.
Lu F, Bytautiene E, Tamayo E, et al. Gender-specific effect of
overexpression of sFlt-1 in pregnant mice on fetal programming of
blood pressure in the offspring later in life. Am J Obstet Gynecol.
2007; 197: 418.e1 5.
Lu F, Longo M, Tamayo E, et al. The effect of over-expression of
sFlt-1 on blood pressure and the occurrence of other manifestations
of preeclampsia in unrestrained conscious pregnant mice. Am J
Obstet Gynecol. 2007; 196: 396.e1 7.
Li Z, Zhang Y, Ying Ma J, et al. Recombinant vascular endothelial
growth factor 121 attenuates hypertension and improves kidney
damage in a rat model of preeclampsia. Hypertension. 2007; 50:
686 692.
Redman CW, Sargent IL. Preeclampsia and the systemic
inflammatory response. Semin Nephrol. 2004; 24: 565 570.
Redman CW, Sargent IL. Latest advances in understanding
preeclampsia. Science. 2005; 308: 1592 1594.
Zhou CC, Zhang Y, Irani RA, et al. Angiotensin receptor agonistic
autoantibodies induce pre-eclampsia in pregnant mice. Nat Med.
2008; 14: 855 862.

Hypertens Res Pregnancy 2013; 1: 5765