243
Biol Res 31: 2 4 3 - 2 5 0 (1998)
Role of nitric oxide in maternal hemodynamics
and hormonal changes in pregnant rats
SOFIA P SALAS*
Center for Medical Research, and Department of Obstetrics and Gynecology,
School of Medicine, Pontifical Catholic University of Chile, Santiago, Chile
Normal pregnancy
is characterized
by a significant
reduction
in total
peripheral
vascular resistance and decreased pressor responsiveness
to
vasodilator
agents. This review will consider whether nitric oxide (NO)
contributes to these changes, and whether a deficiency of NO produces a pre­
eclampsia like syndrome. The biosynthesis
of NO increases in pregnant
animals, as assessed by the raised plasma concentration,
urinary
excretion
and metabolic production
rate of guanosine 3',5'-cyclic
monophosphate
(cGMP), the second messenger of NO. In addition, urinary excretion of nitrate,
the stable metabolites of NO, increases during pregnancy, paralleling the rise
in cGMP. Several studies provide convincing
evidence indicating
that
expression and activity of different NO synthases (NOS) are increased in
gravid animals. Acute blockade of NOS causes a dose response increase in
blood pressure
and reverses
the blunted
vasopressor
response
to
vasoconstrictor
agents. Long-term NOS inhibition produces a
pre-eclampsia
like syndrome,
characterized
by maternal
hypertension,
proteinuria,
thrombocytopenia,
and renal damage, and lower litter size and fetal weight.
Both acute and chronic responses are reduced when L-arginine, the substrate
for NOS, is administered
in high doses, indicating that these changes are
specific
to NO inhibition.
In conclusion,
present data suggest that a
disturbance
in NO release may contribute
to the pathogenesis
of pre­
eclampsia.
Key terms: fetal growth, maternal hemodynamics,
nitric oxide, nitric
synthase, pre-eclampsia like syndrome, rat pregnancy.
INTRODUCTION
Normal pregnancy is characterized by a
marked
stimulation
of the
reninangiotensin-aldosterone system (RAAS),
which c a u s e s renal w a t e r and s o d i u m
retention, thus increasing plasma volume
(Longo, 1983; Wilson et al, 1980). Plasma
volume expansion is essential for normal
fetal g r o w t h , as it allows a sustained
oxide
elevation in cardiac output and, indirectly,
in uterine blood flow. Despite the rise in
blood volume and the activation of the
vasoconstrictor RAAS, blood pressure does
not increase during pregnancy (Wilson et
al, 1980). At least two facts account for the
normal blood pressure during pregnancy:
there is a significant reduction in total
peripheral vascular resistance and pressor
responsiveness to vasoconstrictor agents is
C o r r e s p o n d e n c e to: Dr Sofía P Salas, Centro de Investigaciones Médicas, Escuela de Medicina, Pontificia Universidad
Católica de Chile, Casilla 114-D, Santiago 1, Chile. Phone: (56-2) 6 3 2 - 5 9 4 0 . Fax: (56-2) 6 3 2 - 1 9 2 4 . E-mail:
ssalas @ med.puc.cl
244
Biol Res 31: 2 4 3 - 2 5 0 (1998)
decreased (Gant et al, 1973; Phippard et al,
1986). T h e r e n a l v a s c u l a r b e d a l s o
participates in this vasodilatory response to
pregnancy. Despite the decline in mean
arterial blood pressure (MAP), renal blood
flow and glomerular filtration rate (GFR)
increase about 50% above preconception
levels. These changes underline the marked
fall in renal vascular resistance that occurs
during pregnancy (Conrad, 1984). The
mechanisms
responsible
for
these
hemodynamic changes during pregnancy
are still controversial.
In recent years, the role of nitric oxide
(NO) as a potent vasodilator has become well
accepted. The purpose of the present review
is to provide evidence that NO participates in
the cardiovascular adaptation to normal
pregnancy. We will specifically discuss the
changes in nitric oxide-nitric oxide synthase
system in pregnant rats because this species
offers many advantages. Cardiovascular and
renal changes during rat pregnancy, such as
increased uterine blood flow, decreased
MAP, attenuation of pressor responsiveness
to exogenous vasoconstrictor agents, and
increased GFR are similar to those observed
in pregnant women. In addition, rats are
relatively inexpensive and easy to breed,
making them suitable for their use in
research.
BIOLOGY OF NITRIC OXIDE
Nitric oxide is an inorganic free radical gas
that has, among other known functions,
important cardiovascular effects related to
its ability to inhibit platelet aggregation and
relax vascular and uterine smooth muscle. It
has a half-life of a few seconds and in
biological systems it decomposes rapidly to
nitrite and nitrate. NO is synthesized from
L - a r g i n i n e by different n i t r i c o x i d e
synthases (NOS), and L-citrulline is the
byproduct (Vallance & Collier, 1994). So
far, three isoforms of NOS have been
identified: a neuronal type (nNOS or NOS
I), a macrophage or inducible type (iNOS or
NOS II), and an endothelial type (eNOS or
NOS III). The neuronal isoform is found in
central and peripheral neurons, macrophage
type NOS is expressed after activation of
cells with certain inflammatory mediators,
and e n d o t h e l i a l i s o f o r m is p r e s e n t in
vascular endothelium, platelets and heart
(Ignarro, 1991). NO produced in endothelial
cells rapidly diffuses out into nearby target
cells, such as vascular smooth muscle and
blood platelets, where N O activates the
soluble form of guanylate cyclase to raise
the intracellular levels of guanosine 3 ' , 5'cyclic monophosphate (cGMP). Along with
this p a r a c r i n e r o l e , N O may have an
autocrine function by inhibiting the activity
of NOS in endothelial cells (Fig 1).
Due to its short half-life, it has been
difficult to measure NO directly. Therefore,
researchers have used different strategies to
determine N O - N O S activation, such as:
measurement of NOS enzyme activity by
using the [ H]-citrulline conversion assay;
i m m u n o h i s t o c h e m i c a l l o c a l i z a t i o n of
d i f f e r e n t N O S i s o f o r m s ; d e t e c t i o n of
3
Nitric Oxide - NOS Pathway
Vascular Endothelial Cell
L-Arginlne
N O - A RG
L - NAME
L-Cltrulllne,
NO
Vascular S m o o t h Muscle
Blood Platelet
F i g 1. S c h e m a t i c representation of the transcellular
mechanisms by which NO communicates with nearby
target cells. A vascular endothelial cell that generates NO
from L-arginine is represented. NO diffuses out of the
endothelial cell into vascular smooth muscle cells and
blood platelets, where NO activates cytosolic guanylate
cyclase to raise intracellular levels of cGMP. In addition,
NO may have an autocrine function by inhibiting NOS in
its endothelial cell of origin. (Adapted from Ignarro, 1991).
245
Biol Res 31: 243-250 (1998)
messenger RNA for NOS; measurement of
nitrite/nitrate, the stable metabolites of NO,
and measurement of c G M P , the second
messenger for NO. In addition, either acute
or chronic administration of structural
analogs of L-arginine, such as Nco-nitro-Larginine or nitro-L-arginine methyl ester
( L - N A M E ) , c o m p e t i t i v e l y i n h i b i t the
formation of nitric oxide, which can be
reversed by excess amounts of L-arginine,
thus providing a valuable tool to study the
role of NO in different biological systems.
EVIDENCE THAT NO-NOS ARE INCREASED
IN R A T P R E G N A N C Y
Changes in cGMP and in
nitrite-nitrate
excretion
Results available in the literature strongly
suggest that production of endogenous NO
is increased in gravid rats. Plasma levels
and urinary excretion rates of cGMP are
i n c r e a s e d d u r i n g rat g e s t a t i o n . T h e s e
findings most probably reflect increased
tissular production of cGMP. A metabolic
study d e m o n s t r a t e d increased entry of
cGMP into the plasma compartment, rather
than d e c r e a s e d c l e a r a n c e ( C o n r a d &
Vernier, 1989). Interestingly, pseudopregnant rats also exhibit enhanced urinary
c G M P e x c r e t i o n , s u g g e s t i n g that the
proliferative activity that accompanies feto­
placental maturation, as well as placental
hormones, are not necessary for the rise in
urinary excretion of cGMP. Although these
findings are considered as indirect evidence
of increased NO-NOS activity during rat
p r e g n a n c y and p s e u d o p r e g n a n c y , it is
worth noting that other mediators, such as
atrial natriuretic peptide, may also produce
e l e v a t i o n s of c G M P and v a s o d i l a t i o n
during pregnancy.
T h e u r i n a r y e x c r e t i o n and p l a s m a
levels of the stable N O metabolite nitrate
are increased in pregnant and p s e u d o p r e g n a n t r a t s , p a r a l l e l i n g the r i s e in
urinary c G M P excretion. Chronic treat­
ment with L-NAME inhibits the increase
in urinary nitrate excretion, indicating that
it is a consequence of NO activity (Conrad
etal, 1993).
Nitric oxide synthases during
pregnancy
Several studies provide convincing evidence
indicating that NOS expression and activity
i n c r e a s e during p r e g n a n c y . The gene
expression of endothelial constitutive nitric
oxide synthase is elevated in rat aorta during
pregnancy. Estradiol supplementation to
gonadectomized rats reproduces this change,
w h e r e a s p r o g e s t e r o n e and t e s t o s t e r o n e
administration has no effect (Goetz et al,
1994). Another study showed increased levels
of calcium-dependent NOS activity in uterine
artery, heart, kidney and skeletal muscle
obtained from near-term pregnant Guinea
pigs. These changes were also mimicked
when ovariectomized animals were treated
with estradiol, thus supporting the idea that
the increment in NOS activity is under
estrogen influence (Weiner et al, 1994).
Nerve fibers containing NOS have been
localized in mid-term pregnant rat uterus; the
greatest number of NOS positive nerve fibers
was localized near the cervix, whereas
endothelial cell NOS activity appeared to
distribute uniformly. Interestingly, the
presence of NOS activity in the pregnant rat
uterus declined near term, as shown by
histochemical and biochemical assays
(Natuzzi et al, 1993). Another study
demonstrated the presence of iNOS staining in
cells at the fetal-maternal interface of the rat
placenta; this staining was greatly reduced
during labor (Purcell et al, 1997). In addition,
using i m m u n o h i s t o c h e m i c a l techniques,
Riemer and coworkers (1997) revealed the
expression of two NOS isoforms in the
pregnant rat uterus: eNOS was localized in the
vascular endothelium, and iNOS in the
myometrial and vascular smooth muscles, as
well as in the decidual epithelium; the
expression of both isoforms declined
significantly in laboring rats. Similarly, NOS
activity, evaluated by measuring the difference
in radiolabeled arginine to citrulline
conversion, decreased between days 15 and 21
of gestation (Sladek & Roberts, 1996). The
changes in placental and uterine NOS
expression and activity suggest a paracrine
role for NO in regulating uterine contractility,
blood flow and immunosuppression required
for pregnancy maintenance. NO withdrawal at
term may also be involved in the initiation of
246
Biol Res 31: 243-250 (1998)
labor. A brief summary of the evidence
indicating increased NO biosynthesis during
pregnancy in rats is provided in Table I.
MATERNAL AND FETAL EFFECTS OF ACUTE
AND LONG-TERM NOS INHIBITION
Maternal effects of acute NOS
inhibition
Acute administration of L-NAME or N00n i t r o - L - a r g i n i n e to c o n s c i o u s
rats
produces a dose-response increase in MAP
of s i g n i f i c a n t l y g r e a t e r m a g n i t u d e in
pregnant rats in late gestation than in
either non-pregnant rats, or in pregnant
r a t s in m i d g e s t a t i o n ( M o l n a r &
Hertelendy, 1992; Nathan et al, 1995).
This response is abolished by L-arginine
administration, providing strong evidence
that the action of this inhibitor is due
s p e c i f i c a l l y to t h e i n h i b i t i o n of N O
synthesis from L-arginine. In addition,
continuous infusion of NOS inhibitors to
pregnant rats reverses the blunted
v a s o p r e s s o r r e s p o n s e to angiotensin II
(ANG II), vasopressin, and norepinephrine
obtained during gestation, suggesting that
N O is i n v o l v e d
in t h e
vascular
refractoriness observed during pregnancy
(Molnar & Hertelendy, 1992).
The renal circulation also participates in
the vasodilatory response to pregnancy.
Baseline GFR and effective renal plasma
flow are s i g n i f i c a n t l y i n c r e a s e d , and
e f f e c t i v e r e n a l v a s c u l a r r e s i s t a n c e is
d e c r e a s e d in c h r o n i c a l l y i n s t r u m e n t e d
g r a v i d r a t s , as c o m p a r e d with v i r g i n
c o n t r o l s . D u r i n g i n f u s i o n of N O S
inhibitors, these three parameters equalized
in the pregnant and virgin rats, suggesting
that pregnant animals are more responsive
to N O S i n h i b i t i o n t h a n v i r g i n rats
(Danielson & Conrad, 1995).
In summary, the data from acute studies
s u g g e s t that N O c o n t r i b u t e s to the
attenuated pressor responses of vasocon­
strictor agents, as well as to the renal
vasodilatation and hyperfiltration observed
during pregnancy.
Maternal effects of long-term
inhibition
NOS
Different researchers have investigated the
effects of p r o l o n g e d N O inhibition on
maternal hemodynamics and fetal growth,
by t r e a t i n g p r e g n a n t r a t s with N O S
inhibitors, either diluted in their drinking
solution (Baylis & Engels, 1992; Diket et al,
1994; Pierce et al, 1995; Salas et al, 1995),
given by continuous endovenous infusion
( M o l n á r et al, 1994) or by o s m o t i c
m i n i p u m p s placed subcutaneously (Buhimschi et al, 1995; Helmbrecht et al, 1996;
Yallampalli & Garfield, 1993). Results
obtained by these different routes are quite
comparable, and will be discussed together.
Chronic administration of NOS inhibitors
to gravid rats results in a dose-dependent
Table I
Evidence of increased nitric oxide biosynthesis during pregnancy
Observations
References
Increased plasma cGMP levels
Conrad & Vernier, 1989
Increased urinary cGMP excretion
Conrad & Vernier, 1989
Increased metabolic production rate of cGMP
Conrad & Vernier, 1989
Conrad et al, 1993
Increased urinary excretion and plasma levels of nitrite/nitrate
Conrad et al, 1993
Increased expression of eNOS mRNA in aorta
Goetz et al, 1994
Increased NOS activity in different tissues
Weiner et al, 1994
NOS expression in uterus and placenta changes towards parturition
Natuzzi et al, 1993
Purcell etal.
1997
Riemer et al, 1997
247
Biol Res 31: 2 4 3 - 2 5 0 (1998)
increase in systemic blood pressure in all but
one study (Diket et al, 1994), thus confirming
the major role of NO in the maintenance of
normal vascular tone during pregnancy
(Baylis & Engels, 1992; Molnar et al, 1994;
Salas et al, 1995). An excess of L-arginine,
but not of the inactive stereoisomer Darginine, reduced L-NAME effects on blood
pressure (Buhimschi et al, 1995).
M a r k e d p r o t e i n u r i a and t h r o m b o c y ­
t o p e n i a , and r e d u c e d p l a s m a v o l u m e
expansion were also observed after NOS
inhibition (Baylis & Engels, 1992; Molnar et
al, 1994; Salas et al, 1995; Yallampalli &
Garfield, 1993). Long-term Nco-nitro-Larginine administration to pregnant rats
produced a dose-dependent decrease in
p l a s m a renin a c t i v i t y ( P R A ) w i t h o u t
c h a n g e s in e i t h e r s e r u m or u r i n a r y
aldosterone levels (Salas et al, 1995, 1997)
(Fig 2). Because NO synthesis inhibition
increases blood pressure and consequently
withdraws sympathetic activity (both renin
inhibitory signals), the reduced PRA levels
observed in this condition might be an
indirect consequence of the hemodynamic
changes induced by NO synthesis blockade.
To address this issue, control rats were
instrumented with an intra-aortic balloon
catheter (to control renal perfusion pressure)
and pretreated with propranolol (to eliminate
beta-adrenergic effect). These rats exhibited
an increased PRA in response to L-NAME
treatment (Sigmon et al, 1992). Direct
influence of nitric oxide on renin release
was also explored in an isolated perfused
juxtaglomerular apparatus preparation, in
which influences from both the baroreceptor
and the sympathetic nervous system are
eliminated. In this preparation, addition of
Noi-nitro-L-arginine to the external bath
fluid increased renin release. When the
inhibitor was administered to the luminal
fluid at the macula densa, renin secretion
was decreased by making it less sensitive to
the stimulatory effect of a low luminal NaCl
concentration (He et al, 1995).
Three different studies report no change
in either urinary or serum aldosterone
levels after NO synthesis blockade, despite
marked reductions in PRA (Arnal et al,
1992; Salas et al, 1995; Salazar et al, 1992).
The possible mechanisms of this apparent
dissociation between PRA and aldosterone
levels need to be further explored.
Although NO stimulates
soluble
guanylate cyclase activity, NOS blockade
did not cause significant reductions in
cGMP. cGMP levels, measured in amniotic
fluid, did not display a dose-dependent
reduction with L - N A M E ( D i k e t et al,
1994). Similarly, urinary cGMP excretion
was not reduced either in non-pregnant or
pregnant rats, or in rats treated with NOS
inhibitors (Arnal et al, 1992; Salas et al,
1997) (Fig 3), suggesting that cGMP levels
30
30-
Plasma Volume
PRA
20 i
*
( ng Ang Vml/n )
10-
Aldosterone
( nmol/l ) 4
1.7
0.69
Control
NO-Arg
(mmol/L)
F i g 2 . E f f e c t s on p l a s m a v o l u m e , PRA and serum
aldosterone levels of long-term administration of two
doses of Nco-nitro-L-arginine to pregnant rats from days 7
to 21 of gestation. Data are presented as means + SEMs. *
P < 0.05 vs control group by A N O V A . (Adapted from Sa­
las et al, 1995).
248
Biol Res 31: 243-250 (1998)
Urinary c G M P
100 n
F l a (6-keto-PGFla) excretion (Fig 3); in
consequence, the 6-keto-PGFla/TxB ratio
increased (Salas et al, 1997). In vitro studies
have demonstrated that N O produced by
endothelial cells increased the production of
6-keto-PGFlot and TxB , through activation
of prosta-glandin H synthase, and that LN A M E significantly diminished prosta­
glandins production (Davidge et al, 1995).
Whether this is the mechanism involved in
our observations remains to be elucidated.
As r e p o r t e d in a c u t e e x p e r i m e n t s ,
c h r o n i c N O b l o c k a d e i n c r e a s e d renal
vascular resistance and reduced GFR in
near term pregnant rats (Baylis & Engels,
1992; Molnar et al, 1994). In addition to an
abnormal renal function, chronic NO
blockade produced renal histological
abnormalities, such as focal glomerular
sclerosis, occlusion of glomerular capillary
l u m e n s by e o s i n o p h i l i c m a t e r i a l , and
mesangial cell proliferation with preserva­
tion of the epithelial foot processes. The
proportion of abnormal-appearing glome­
ruli was reduced with the addition of Larginine (Helmbrecht et al, 1996). A mild
diffuse interstitial edema and infiltrate of
lymphocytes were also observed. Although
Molnar et al (1994) reported that this
alteration is unique to pregnancy, other
authors, including us (unpublished data),
have also observed renal damage in virgin
rats (Baylis et al, 1992). This discrepancy
may have been related to the early and
more prolonged exposure of virgin rats to
NOS inhibitors in the latter studies.
Despite the evidence suggesting that NO
might be involved in maintaining uterine
quiescence during pregnancy, chronic NO
i n h i b i t i o n did not a l t e r the day of
spontaneous delivery, thus suggesting that
o t h e r m e c h a n i s m s are r e s p o n s i b l e for
c o n t r o l l i n g the l e n g t h of p r e g n a n c y
( M o l n a r et al, 1 9 9 4 ; Y a l l a m p a l l i &
Garfield, 1993).
2
80
60
(nmol/24h)
40
2
20
0
Urinary 6-keto-PGF1 a
200
150
(ng/24h)
1
0
0
50
U r i n a r y T h r o m b o x a n e B2
120 n
100
*
80
(ng/24h)
g,,
40
20
0
NO-Arg
Control
Fig 3 . Effects on urinary excretion of cGMP, 6-ketoP G F l a and thromboxane B2 of long-term administration
of 1.7 mmol/L of Nco-nitro-L-arginine to pregnant rats
from days 7 to 21 of gestation. Data are presented as
means + SEMs. * P < 0.05 vs control group by unpaired
Student's t-test. (Adapted from Salas et al, 1997).
may be i n f l u e n c e d by a c t i v a t o r s of
particulate guanylate cyclase, such as atrial
natriuretic peptide (ANP). In this respect, it
is worth noting that the administration of LNAME to non-pregnant rats increased basal
ANP levels and enhanced stretch-induced
ANP release (Leskinen et al, 1995).
Chronic NOS blockade also influences
maternal levels of other vasoactive agents.
Pregnant rats treated with Nco-nitro-Larginine e x h i b i t e d a reduced urinary
excretion of thromboxane B (TxB ), without
significant changes in 6-keto-prostaglandin
2
2
Fetal effects of long-term
NOS
inhibition
Chronic NO blockade, in a dose-dependent
manner, caused a significant reduction in
the weight and size of the pups, as well as
reduced litter size and increased fetal
mortality (Baylis & Engels, 1992; Salas et
Biol Res 31: 2 4 3 - 2 5 0 (1998)
al, 1995; Yallampalli & Garfield, 1993).
We have proposed that the mechanisms
causing fetal growth retardation are related
to altered maternal vasodilation, which
limits p l a s m a v o l u m e e x p a n s i o n and,
secondarily, reduces cardiac output and
utero-placental blood flow. Nevertheless, a
direct role of NO deficiency in placental
perfusion cannot be excluded, since it is
well k n o w n t h a t the p l a c e n t a l v i l l u s
v a s c u l a r t r e e h a s t h e a b i l i t y to b o t h
generate and respond to NO (Myatt et al,
1991). A progressive and dose dependent
hind limb d i s r u p t i o n in the pups was
observed after NO blockade (Diket et al,
1994; Salas et al, 1995). This alteration
corresponded to hemorrhagic necrosis, with
marked cellular infiltration and loss of
s t r u c t u r e . It w a s r e v e r s e d with the
administration of the nitric oxide donor
sodium nitroprusside or with an excess of
L-arginine. When dams were treated with
the inactive enantiomer, D-NAME, none of
the pups presented with hind limb defects.
These results strongly suggest that hind
limb disruption is specifically related to
NO synthesis inhibition (Diket et al, 1994;
Pierce et al, 1995). Most probably, this
alteration is caused in the post-organogenic
period since it is not produced when the
drug is given during the first week of
pregnancy (Salas et al, 1995). In addition,
no defect was observed when the dams
received aminoguanidine, a selective
inhibitor of iNOS (Pierce et al, 1995),
providing evidence supportive of specific
i n h i b i t i o n of e n d o t h e l i a l ( c N O S ) N O
release in the pathogenesis of this defect.
The finding that lesions induced by
chronic inhibition of NO synthesis, such as
maternal hypertension, proteinuria, and renal
glomerular injury and fetal growth restriction
are reversed by treatment with L-arginine
lends support to the potential use of NO
donors in the treatment and prevention of pre­
eclampsia. Nevertheless, the administration
of the endogenous NO synthase-independent
NO donor, molsido-mine, to pregnant rats
treated with L - N A M E , u n e x p e c t e d l y
worsened pregnancy outcome by increasing
fetal reabsorption and reducing litter size and
fetal weight (Richer et al, 1996). Thus, data
available provide a caveat regarding the use
249
of this NO donor in pregnant women with
pre-eclampsia.
OVERVIEW
There is strong evidence that suggests that
increased N O synthesis occurs during
pregnancy and that nitric oxide-nitric oxide
synthases pathway is involved in maintenance
of vascular tone and altered vascular responses
to pressor agents during pregnancy.
Taking altogether, present data may
have important clinical implications. Gant
et al (1973) have shown that the blunting of
the A N G II r e s p o n s e in p r e g n a n c y is
reversed in pregnant women destined to
develop preeclampsia. In addition, NO is
also increased in normal human pregnancy
and seems to be decreased in women with
pre-eclampsia. Moreover, umbilical arteries
and veins from patients with pregnancyinduced hypertension had a significantly
l o w e r r e l e a s e of e n d o t h e l i u m - d e r i v e d
r e l a x i n g f a c t o r than t i s s u e s
from
normotensive pregnant women (Pinto et al,
1991). In addition, inhibition of nitric oxide
synthesis in rats during pregnancy produces
signs similar to those of p r e e c l a m p s i a
( Y a l l a m p a l l i & G a r f i e l d , 1993). It is,
therefore, possible that a disturbance in NO
release, either primary or secondary to
endothelial dysfunction, may contribute to
the pathogenesis of pre-eclampsia.
ACKNOWLEDGEMENTS
This work was supported by grants 1940603 and 196-0779 from the National Fund
for
Scientific
and
Technological
Development (FONDECYT), Chile.
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