From the 2nd Institute of Obstetrics and Gynecology (R. Di I., E.M.,
B.V., E.V.C.), the Department of Internal Medicine (C.L.), and the Department
of Pathology and Experimental Medicine (P.A.), University "La
Sapienza," Rome, Italy.
Correspondence to Romolo Di Iorio, MD, PhD, Laboratory of Perinatal Medicine and Molecular Biology, 2nd Institute of Obstetrics and Gynecology, University "La Sapienza," Viale Regina Elena, 324, I-00161 Rome, Italy. E-mail perinat{at}flashnet.it
During normal pregnancy, important physiological
adaptations occur in the mother that ensure an adequate blood supply to
the fetus. Vascular resistance, mean arterial pressure, and
sensitivity to endogenous constrictors are reduced, whereas
cardiac output, heart rate, and blood volume are
increased.2 This allows the maintenance
of the placental vasculature in a state of near-maximal dilatation.
Failure to achieve these adaptations may result in the reduced
fetoplacental perfusion that develops during disease states such as
preeclampsia and intrauterine growth retardation. Because placenta
lacks autonomic innervation, uteroplacental perfusion is regulated
mainly by systemic blood pressure changes through the action of both
circulating and locally released vasoactive
agents.3 The role of endothelial
cells in the modulation of vascular tone has been extensively
demonstrated, and abnormal endothelial function in
preeclampsia could contribute to an increase in peripheral
resistance.4
Adrenomedullin is a novel hypotensive peptide first isolated from human
pheochromocytoma eliciting a long-lasting vasorelaxant
action.5 Adrenomedullin immunoreactivity and mRNA
have been demonstrated in a number of human and animal tissues,
including rat uterus.6 7 Cultured
endothelial cells secrete adrenomedullin and possess
specific receptors for this peptide.8 9 It has
been demonstrated that adrenomedullin exerts a natriuretic
action on kidneys and peripheral vasculature to control
fluid and electrolyte homeostasis10 11 and
affects angiotensin II secretion. Recently it has been
reported that patients with hypertension show higher levels of plasma
adrenomedullin compared with normotensive control subjects, supporting
the hypothesis that adrenomedullin could participate in the
physiological regulation of blood pressure and
vascular homeostasis.12 We recently have found
high concentrations of adrenomedullin in full-term
pregnancy13 and mRNA for adrenomedullin has been
detected in fetal membranes,14 suggesting a
potential role of this peptide in human reproduction.
In the present study, we investigated the possibility that
adrenomedullin may be involved in the pathophysiology of preeclampsia
and evaluated its concentrations and localization in maternal and
fetoplacental compartments.
In all subjects, plasma was collected from the cubital vein; in
pregnant women, we also obtained amniotic fluid samples by
transabdominal amniocentesis or at elective cesarean section. In some
patients, umbilical cord plasma samples and placenta specimens were
collected at delivery (preeclamptic, n=11; normotensive, n=13).
This study was approved by the local ethics committee, and informed
consent was obtained from all participants.
Sample Collection
In pregnant subjects, maternal blood samples were drawn from the
cubital vein at the time of amniotic fluid sampling and just before the
induction of anesthesia in patients undergoing cesarean
section. In 7 preeclamptic patients, blood samples were also collected
48 hours after delivery. In nonpregnant women, blood samples were
collected in the first half of the ovarian cycle, and none of
the subjects were using oral contraceptives.
At delivery, the umbilical cord was clamped before any signs of
breathing were seen; blood was drawn from the umbilical vein. Blood
samples, anticoagulated with EDTA and aprotinin, were kept on ice until
centrifugation and then were stored at -80°C. Within
10 minutes from delivery, placentas (beginning at 5 cm from the
placental disk) were immediately collected and processed for
immunohistochemistry.
Adrenomedullin Determination
Immunohistochemistry
Statistics
Nonpregnant women with hypertension had significantly
(P<0.01) higher plasma adrenomedullin concentrations
(3.5±0.4 fmol/mL) compared with normotensive nonpregnant women
(1.7±0.2 fmol/mL; Figure 1
No correlation was found between amniotic fluid or umbilical plasma
adrenomedullin values and gestational age, placental weight, or birth
weight in the normotensive and preeclamptic groups.
In placenta, adrenomedullin staining was localized primarily on the
extravillous trophoblast cells, as confirmed using cytokeratin in
consecutive sections (Figure 3A
The mechanism for increased local production of adrenomedullin
in preeclampsia is not known. Amniotic fluid and umbilical vein
adrenomedullin concentrations may reflect either fetal or placental
production. Adrenomedullin is localized in extravillous
trophoblast and endothelial cells of primary villi,
suggesting that placenta is a site of production or binding
during pregnancy. Although immunohistochemical analysis is not
a quantitative technique, we failed to demonstrate any significant
differences in either the percentage and intensity of adrenomedullin
staining between preeclamptic and normotensive placentas. This finding
could indicate that increased adrenomedullin levels in fetoplacental
compartments of preeclamptic pregnant women are not derived from the
placenta. Alternatively, increased adrenomedullin concentrations in
preeclampsia can be explained on the basis of an active secretion of
this peptide by the fetus. Actually, endothelial cells
from the umbilical vein secrete adrenomedullin in culture
systems8 and possess specific adrenomedullin
receptors.9 Furthermore, adrenomedullin has been
implicated in the regulation of endocrine secretion. Adrenomedullin
inhibits the secretion of ACTH,21
aldosterone,22 and
insulin,23 and its secretion is stimulated by
thyroid hormones, progesterone, and
dexamethasone.24 It has been reported
that fetuses from preeclamptic women have higher concentrations of
corticotropin-releasing hormone, ACTH, and
cortisol.25 26 Thus, it is possible that
adrenomedullin secretion is modulated by these factors and may be
involved in the regulation of fetal hypothalamus-pituitary-adrenal
gland response to uteroplacental insufficiency.
We can only speculate on the significance of increased adrenomedullin
levels found in preeclampsia. Adrenomedullin secreted by the fetus
through amniotic fluid and umbilical vein blood may exert its action on
placental cells, inducing the release or inhibition of other vasoactive
peptide output, such as NO or ET-1, thus participating in the
regulation of vascular tone in uteroplacental and fetal circulation. In
preeclampsia, we reported that amniotic fluid concentrations of
ET-115 and NO27 are
increased compared with those in normotensive pregnancies, and
increased NO levels are necessary to maintain an adequate blood flow
through the placenta.18 28 We speculate that in
preeclampsia, increased local production of adrenomedullin may
compensate for the increased synthesis and release from the injured
endothelium of other vasoactive substances such as
thromboxane A2 and ET-1, which act as
vasoconstrictors on placental vasculature. Thus, increased
adrenomedullin concentrations may be necessary to maintain placental
vascular resistance and/or fetal circulation at a
physiological level. Alternatively, this new
vasoactive peptide may have a preeminent role in the regulation of
fetal response to a compromised intrauterine environment, acting on
fetal endocrine secretion.
Received January 15, 1998;
first decision February 9, 1998;
accepted June 26, 1998.
© 1998 American Heart Association, Inc.
Scientific Contributions
Adrenomedullin, a New Vasoactive Peptide, Is Increased in Preeclampsia
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Abstract—Adrenomedullin is a
novel peptide that elicits a long-lasting vasorelaxant activity.
Recently, we found high concentrations of adrenomedullin in maternal
and umbilical cord plasma and in amniotic fluid in full-term
human pregnancy, indicating a role of this peptide during gestation. To
investigate the possibility that adrenomedullin is involved in the
pathophysiology of preeclampsia, we measured its concentration in
maternal and fetoplacental compartments. We studied 12 normotensive
nonpregnant women, 13 hypertensive nonpregnant subjects, 29 patients
with preeclampsia, and 30 normotensive pregnant women. In all patients,
plasma was collected from the cubital vein, and amniotic fluid samples
were obtained by transabdominal amniocentesis or at elective cesarean
section. Plasma samples from umbilical vein and placental tissues were
collected at delivery. Adrenomedullin was assayed on plasma and
amniotic fluid samples using a specific radioimmunoassay, and its
localization and distribution on placental sections was determined by
immunohistochemistry. Adrenomedullin concentrations were higher in
hypertensive than in normotensive nonpregnant patients. Pregnant women
had higher adrenomedullin levels than nonpregnant subjects, although
maternal plasma adrenomedullin concentrations did not differ between
normal pregnant and preeclamptic women. Preeclamptic patients showed
higher concentrations (P<0.01) than normotensive
pregnant women of adrenomedullin in amniotic fluid (252±29 versus
112±10 fmol/µmol creatinine) and umbilical vein plasma
(18.1±2.1 versus 8.5±1.1 fmol/mL). Increased local production
of adrenomedullin is associated with preeclampsia. The fetus seems to
be responsible for the higher levels of this hormone. Increased
adrenomedullin concentrations may be necessary to maintain placental
vascular resistance and/or fetal circulation at a physiological
level.
Key Words: adrenomedullin • preeclampsia • placenta • amniotic fluid • plasma
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Preeclampsia is a serious and common complication of
pregnancy, and despite several research efforts, its pathogenesis is
still unclear. It has been suggested that failure of trophoblast
invasion of the maternal placental bed may be responsible for impaired
placental perfusion, causing the release of cytotoxic factors
that cause maternal endothelial damage and dysfunction
in a variety of organs.1
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Patients
We examined 4 groups of women: (1) 12 normotensive, healthy
nonpregnant women (aged 23 to 36 years), (2) 13 hypertensive (WHO stage
I) nonpregnant women (aged 25 to 41 years), (3) 29 pregnant women with
preeclampsia at 25 to 38 weeks of gestation (aged 21 to 37 years), and
(4) 30 normotensive patients (aged 20 to 38 years) matched for
gestational age. Preeclampsia was defined as elevated blood pressure
(>140/90 mm Hg) in at least 4 measurements taken 6 hours apart,
in association with proteinuria (>0.3 g/d), appearing at a gestational
age of >20 weeks. Patients with intrauterine growth retardation
associated with hypertension were excluded from the study, as were
pregnant women with preexisting hypertension, diabetes, or multiple
gestation. None of the pregnant women were in labor at the time of
study.
Samples of amniotic fluid were collected by transabdominal
amniocentesis or by transuterine amniocentesis at the time of elective
cesarean section. In preeclamptic patients, the indication for
amniocentesis was fetal lung maturity assessment (16 cases). In the
control group, amniocentesis was performed to exclude a fetal infection
by cytomegalovirus (polymerase chain reaction technique) suspected on
the basis of maternal serology (11 cases). In these patients, prenatal
diagnosis and neonatal outcome were negative for the suspected
infection. None of the patients experienced uterine
contractility before cesarean section. Indications for
elective cesarean section in the normotensive group included breach
presentation, previous cesarean section, premature rupture
of membranes in absence of labor, placenta previa, and maternal cardiac
disease. In patients undergoing transabdominal amniocentesis, the
initial 3 mL of amniotic fluid was collected and processed. In women
undergoing elective cesarean section, samples were collected by
transuterine amniocentesis at the time of surgery. Briefly, amniotic
fluid samples were immediately centrifuged after collection at
1800g for 15 minutes at 4°C; the supernatants were divided
into aliquots and stored at -80°C until assayed.
Plasma and amniotic fluid adrenomedullin concentration was
measured after extraction and purification. Briefly, 2 mL of sample was
applied to conditioned Sep-Pak C18 columns (Millipore Corp, Waters
Chromatography), and the column was sequentially
washed with 5 mL of isotonic saline, 5 mL of 0.1% trifluoroacetic
acid, and 5 mL of 20% acetonitrile in 0.1% trifluoroacetic acid. The
absorbed material was eluted with 4 mL of 50% acetonitrile, and the
eluate was lyophilized. After lyophilization, samples were dissolved in
50 mmol/L phosphate buffer (pH 7.4), and adrenomedullin was
measured in plasma and amniotic fluid by radioimmunoassay using a
commercial kit (Phoenix Pharmaceuticals Inc) with rabbit polyclonal
antibody raised against human adrenomedullin 1-52. The antibody
cross-reacts 100% with human adrenomedullin; no cross-reactivity was
reported with rat adrenomedullin, amylin, calcitonin gene–related
peptide, endothelin-1, or 
-atrial natriuretic peptide.
The intra- and interassay coefficients of variance were 5.1% and
12.0%, respectively.
Specimens of placental tissue collected at delivery were fixed
in 4% paraformaldehyde-0.2% gluteraldehyde, washed,
and embedded in paraffin. The presence of adrenomedullin was determined
by immunohistochemistry on 5-µm paraffin sections processed as
reported previously.15 The sections were stained
using the avidin-biotin-peroxidase technique (Vector ABC, Vector
Laboratories) and incubated with polyclonal antibody raised in rabbits
against purified human adrenomedullin 1-52 (Peninsula Laboratories Inc)
at a dilution of 1:600. Negative control tests were conducted on
placental tissue incubated with either nonimmune rabbit serum, antibody
dilution buffer, or the primary antibody preabsorbed with an excess of
human adrenomedullin (1 µmol/L). The number of positive cells
was quantified using a quantitative system (field=0.175
mm2 at x250 magnification). Ten randomly
selected fields were independently counted by 3 different examiners (R.
Di I., E.M., P.A.) by visual examination, and the proportion of stained
cells was expressed as a percentage of the total cells (stained and
unstained). Cells were considered to be positively stained when
a brown granular staining of the cytoplasm was revealed at low-power
magnification (x10).
Adrenomedullin concentrations are expressed as mean±SEM.
Statistical analysis was performed with the determination of
Spearman rank-order correlation and comparison between groups by
Kruskal-Wallis 1-way ANOVA (Dunn's method) for adrenomedullin
concentrations in maternal plasma. The statistical analysis
involved the paired t test of adrenomedullin values between
the predelivery and postdelivery samples in preeclamptic patients. The
Mann-Whitney U test was used for the statistical
analysis of adrenomedullin levels in amniotic fluid and
umbilical vein plasma between normotensive and preeclamptic women,
since data were not normally distributed. To compare proportions of
cells stained positive for adrenomedullin, Fisher's exact test was
used. Clinical characteristics of women are expressed as mean±SD and
were compared by Student's t test for unpaired data.
Statistical significance was set at P<0.05.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Characteristics of the study subjects are reported in the
Table
. Because there were fewer umbilical
blood and placental tissue samples than maternal blood and amniotic
fluid samples, the patient details have been separated according to
different components of the study. In the maternal blood and amniotic
fluid group, there was no significant difference in gestational age at
sampling between normotensive and preeclamptic women; however, the mean
gestational age at delivery was significantly lower in the preeclamptic
group. In the umbilical blood and placental tissues group, gestational
age at sampling corresponded obviously to the gestational age at
delivery, and there was no significant difference between patient
groups.
View this table:
[in a new window]
Table 1. Demographic, Clinical, and Biochemical Features of Studied
Groups ).
Adrenomedullin levels in normotensive pregnant patients (10.4±0.9
fmol/mL) were 5-fold higher than those detected in normotensive
nonpregnant women. Although in preeclamptic patients plasma
adrenomedullin concentrations (11.3±0.9 fmol/mL) were higher compared
with those in normotensive pregnant women, this difference was not
statistically significant. Also, when adjusted for maternal serum
creatinine, adrenomedullin levels did not differ between
normotensive and hypertensive patients, either pregnant or nonpregnant.
After 48 hours from delivery in preeclamptic women, plasma
adrenomedullin concentrations (6.2±1.5 fmol/mL) were significantly
(P<0.05) reduced compared with predelivery values. No
correlations were found between adrenomedullin plasma concentrations
and gestational age in either normotensive or hypertensive pregnant
women. Preeclamptic patients showed higher concentrations
(P<0.01) of adrenomedullin than normotensive pregnant
patients in umbilical vein plasma (18.1±2.1 versus 8.5±1.1 fmol/mL)
and in amniotic fluid (25.6±1.4 versus 17.6±1.4 fmol/mL). To ensure
that differences between groups were not due to a different rate of
fetal urine production, the amniotic fluid measurements of
adrenomedullin were normalized for creatinine as
reported16 ; after normalization, amniotic fluid
adrenomedullin levels were significantly (P<0.01) higher in
preeclamptic patients (252±29 versus 112±10 fmol/µmol
creatinine; Figure 2).
View larger version (39K):
[in a new window]
Figure 1. Adrenomedullin concentrations (mean±SEM) in
plasma from nonpregnant women (NP), nonpregnant hypertensive subjects
(EH), normotensive pregnant women (NT), preeclamptic patients (PE), and
at 48 hours after delivery in PE patients. a indicates
P<0.01 vs NP; b, P<0.01
vs EH; and c, P<0.05 vs PE.
View larger version (20K):
[in a new window]
Figure 2. Adrenomedullin concentrations in amniotic fluid
and umbilical vein in normotensive (white boxes) and preeclamptic (gray
boxes) patients. The lower and upper bars represent the 10th
and 90th centiles, respectively, and the interquartile range is
indicated by the box, with the median value being the horizontal line
in the box. There was a significant difference between groups
(P<0.01) in both amniotic fluid and umbilical plasma
adrenomedullin concentrations (Mann-Whitney U test for
nonparametric values). and 3B),
and in scattered areas of syncytiotrophoblast (Figure 3E), although in
most of the villi these cells appeared negative.
Endothelial cells in the chorionic plate and in the
primary villi vessels stained for adrenomedullin. The intensity and
percentage of cells stained for adrenomedullin in preeclamptic placenta
(Figure 3C and 3F) did not differ from those in sections obtained from
normotensive pregnancies. All the tissues examined showed the same
immunoreactivity pattern, and no staining was found in negative
controls (Figure 3D).
View larger version (164K):
[in a new window]
Figure 3. Immunohistochemical staining of adrenomedullin in
normotensive (B and E) and preeclamptic (C and F) placentas.
Panel A shows cytokeratin staining (1:1000) in consecutive sections.
Positive immunostaining is localized in the
extravillous trophoblast cells (t) in both placentas (B and C) as
confirmed by cytokeratin (A). Scattered areas of syncytiotrophoblast
cells (s) of villi stained for adrenomedullin and
immunostaining was localized in
endothelial cells (e) of villi and placental basal
plate (E and F). No differences were detected in the intensity of
immunostaining and the proportion of positive cells
between groups. Preabsorption of primary antibody with synthetic
peptide (1 µmol/L) shows no immunostaining (D).
Magnification x400.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
This study confirms our previous report on elevated concentrations
of adrenomedullin in pregnant women compared with nonpregnant
women.13 We also found that hypertensive disorder
is associated with higher plasma adrenomedullin concentrations than in
normotensive patients, as reported by others.12
Although maternal adrenomedullin concentrations did not differ
significantly between preeclamptic and normal pregnant women,
adrenomedullin levels were increased in amniotic fluid and umbilical
vein blood collected from preeclamptic pregnant women. In these
patients, 48 hours after delivery, adrenomedullin concentrations
decreased significantly at levels corresponding to those found
postpartum in normal pregnancies.13 These
findings confirm local production of adrenomedullin and suggest
that it may modulate fetoplacental hemodynamics through
a paracrine mechanism interacting with other vasoactive agents in
physiological and pathological states during
pregnancy, such as preeclampsia. Recent studies have shown that the
vasodilatation induced by adrenomedullin is mediated by the release of
nitric oxide (NO)17 and that NO is necessary to
maintain an adequate blood flow through the uteroplacental
circulation.18 Moreover, adrenomedullin has been
demonstrated to inhibit thrombin- and platelet-derived growth
factor–induced endothelin-1 (ET-1) production through a
cAMP-dependent process.19 In the placenta, ET-1
is localized in syncytiotrophoblast and endothelial
cells of villi,20 and there is evidence that this
peptide has a regulatory role in placental
circulation.15
Acknowledgments
This work was supported by the Italian National Research Council
[CNR] (grant 96.01764.CT11).
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
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