Am J Physiol Endocrinol Metab 309: E852–E860, 2015.
First published September 22, 2015; doi:10.1152/ajpendo.00272.2015.
Downregulation of apelin in the human placental chorionic villi from
preeclamptic pregnancies
Liliya M. Yamaleyeva,1 Mark C. Chappell,1 K. Bridget Brosnihan,1 Lauren Anton,1 David L. Caudell,3
Sara Shi,1 Carolynne McGee,1 Nancy Pirro,1 Patricia E. Gallagher,1 Robert N. Taylor,2 David C. Merrill,2
and Heather L. Mertz2
1
Hypertension and Vascular Research Center, Wake Forest School of Medicine, Winston-Salem, North Carolina; 2Department
of Obstetrics and Gynecology, Wake Forest School of Medicine, Winston-Salem, North Carolina; and 3Department of
Pathology/Comparative Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina
Submitted 15 June 2015; accepted in final form 16 September 2015
apelin-13; apelin receptor; ACE2; pregnancy; explant
PREECLAMPSIA IS A pregnancy-induced hypertensive disorder
that occurs in 5–7% of all pregnancies worldwide (54). It is
associated with maternal perinatal morbidity and mortality and
a high risk of premature birth and fetal growth restriction.
Despite continued research, the pathogenesis of preeclampsia
is still unknown. The endogenous apelin system is an emerging
target for the regulation of cardiovascular homeostasis (22);
however, its role in pregnancy is not well understood. The
levels of preproapelin mRNA are widespread in human tissues,
and high levels were identified in the placenta (38), suggesting
Address for reprint requests and other correspondence: L. M. Yamaleyeva, Hypertension and Vascular Research Center, Wake Forest
School of Medicine, Medical Center Blvd., Winston-Salem, NC 271571032 (e-mail: [email protected]).
E852
a possible placental origin of apelin in pregnancy. Apelin may
have a paracrine role in human chorionic villi as it has been
identified in cytotrophoblasts, in syncytiotrophoblasts, and in
fetal endothelial cells (13, 15). A positive correlation between
maternal and fetal plasma apelin was observed in full-term
normal pregnancies (37). Moreover, a transplacental transfer of
apelin with potential impact on fetal growth was suggested
(37). Apelin is a ligand for the human G protein-coupled
receptor APJ (40). The role of APJ in embryonic development
was shown by the existence of cardiovascular developmental
defects in APJ-deficient mice (11). In addition, a small litter
size and a significant loss of homozygous animals were noted
in these animals (11). The apelin/APJ system has pleiotropic
roles in cardiovascular physiology (40). Apelin’s ability to
lower blood pressure was demonstrated chronically in a mouse
model of atherosclerosis and acutely in patients with heart
failure (12, 22, 31, 32). Moreover, apelin induces cardiac
contractility (48) and vessel formation (25) and suppresses
aortic inflammation via downregulation of interleukin-6 and
TNF-␣ mRNA (33). Because of the cardiovascular protective
actions of apelin in addition to its role in the regulation of fluid
homeostasis and metabolic functions, the endogenous apelin/
APJ system may be important for adaptation to pregnancy and
for regulation of fetal growth (51).
The regulation of apelin peptides in the placenta is unknown.
Evidence supports a counterregulatory role for apelin in relation to the renin-angiotensin system (RAS) through the functional interactions between apelin and angiotensin II (ANG II)
(16, 21, 43). ANG II and apelin have opposing actions on
blood pressure regulation, vascular tone, inflammation, and
fluid homeostasis, factors known to be important in pregnancy
(17, 51, 52). Although the systemic RAS is downregulated in
preeclampsia (34), the contribution of local uteroplacental
ANG II/ANG II receptor 1 (AT1R) to the pathogenesis of
preeclampsia has been suggested (1– 4, 7, 17–19). Anton and
Brosnihan (1) demonstrated the upregulation of vasoconstrictor
and proinflammatory peptide ANG II and AT1R in the chorionic villi of women with preeclampsia at term, suggesting a
paracrine role of local ANG II/AT1R in the placenta. Moreover, preeclampsia in female rats with overexpression of human angiotensinogen gene mated to male rats with overexpression of human renin gene (17) exhibits upregulated placental
ANG II, underscoring the contribution of the local uteroplacental RAS to preeclampsia. It is possible that ANG II may
downregulate apelin expression or its release from the preeclamptic chorionic villi and therefore interfere with its action
in preeclampsia.
0193-1849/15 Copyright © 2015 the American Physiological Society
http://www.ajpendo.org
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Yamaleyeva LM, Chappell MC, Brosnihan KB, Anton L,
Caudell DL, Shi S, McGee C, Pirro N, Gallagher PE, Taylor RN,
Merrill DC, Mertz HL. Downregulation of apelin in the human
placental chorionic villi from preeclamptic pregnancies. Am J Physiol
Endocrinol Metab 309: E852–E860, 2015. First published September
22, 2015; doi:10.1152/ajpendo.00272.2015.—The role of the endogenous apelin system in pregnancy is not well understood. Apelin’s
actions in pregnancy are further complicated by the expression of
multiple forms of the peptide. Using radioimmunoassay (RIA) alone,
we established the expression of apelin content in the chorionic villi
of preeclamptic (PRE) and normal pregnant women (NORM) at
36 –38 wk of gestation. Total apelin content was lower in PRE
compared with NORM chorionic villi (49.7 ⫾ 3.4 vs. 72.3 ⫾ 9.8
fmol/mg protein; n ⫽ 20 –22) and was associated with a trend for
lower preproapelin mRNA in the PRE. Further characterization of
apelin isoforms by HPLC-RIA was conducted in pooled samples from
each group. The expression patterns of apelin peptides in NORM and
PRE villi revealed little or no apelin-36 or apelin-17. Pyroglutamate
apelin-13 [(Pyr1)-apelin-13] was the predominant form of the peptide
in NORM and PRE villi. Angiotensin-converting enzyme 2 (ACE2)
activity was higher in PRE villi (572.0 ⫾ 23.0 vs. 485.3 ⫾ 24.8
pmol·mg⫺1·min⫺1; n ⫽ 18 –22). A low dose of ANG II (1 nM; 2 h)
decreased apelin release in NORM villous explants that was blocked
by the ANG II receptor 1 (AT1) antagonist losartan. Moreover,
losartan enhanced apelin release above the 2-h baseline levels in both
NORM and PRE villi (P ⬍ 0.05). In summary, these studies are the
first to demonstrate the lower apelin content in human placental
chorionic villi of PRE subjects using quantitative RIA. (Pyr1)-apelin-13 is the predominant form of endogenous apelin in the chorionic
villi of NORM and PRE. The potential mechanism of lower apelin
expression in the PRE villi may involve a negative regulation of
apelin by ANG II.
APELIN PEPTIDES IN HUMAN PLACENTA IN PREECLAMPSIA
MATERIALS AND METHODS
Human subjects. Group 1, the normal pregnancy group (n ⫽ 22),
consisted of normotensive pregnant subjects (systolic blood
pressure ⬍140/90 mmHg) in their third trimester of pregnancy with
no history of chronic blood pressure elevation and the absence of
proteinuria (4). Group 2, the preeclamptic group (n ⫽ 20), consisted
of women with new-onset hypertension (⬎140/90 mmHg) and proteinuria (ⱖ300 mg/24 h or 2⫹ or more protein on a random sample on
2 or more occasions 4 h apart). Consent forms were signed by all
participants at the time of their enrollment in the study. Systolic and
diastolic blood pressures and proteinuria were recorded at the time of
delivery. All pregnant women were nulliparous and underwent natural
delivery or cesarean section. All subjects were free of any known
preexisting cardiovascular, endocrine, or connective tissue diseases.
The study was approved by the Institutional Review Boards at both
Wake Forest School of Medicine and Forsyth Medical Center (BF04222) (4).
Human placental chorionic villous tissue collection. Immediately
after delivery (within 15 min), the whole placenta was collected on
ice, and tissue sections were taken from the center of the placenta,
near the umbilical cord attachment site. For each tissue section, the
maternal basal plate and fetal membranes were removed so that
only the fetal villous tissue was present; areas of necrosis or tissue
damage were avoided. Each piece of tissue was either 1) snap
frozen in liquid nitrogen and stored at ⫺80°C for future experiments (4), or 2) several small pieces were placed into a cassette and
fixed in 10% neutral buffered formalin for 24 h for immunohistochemistry. The tissue was taken as part of a previously published
study (4); the additional aliquots used for the study were kept
frozen at ⫺80°C until analyzed.
Human placental chorionic villous explant culture. Chorionic villi
were collected from women with normal and preeclamptic pregnancy
using the same criteria as described above. After a 2-h equilibration
period, chorionic villi (1 g of tissue) were exposed to 1 nM of ANG
II alone or in combination with losartan (1 ␮M), an antagonist for
AT1R for 2 h under 5% N2 (PO2 in media ⫽ 60 mmHg) supplemented with 5% CO2 as previously described by our group (45).
The unstimulated release of apelin from chorionic villi for 2 and 16
h was also determined (45). The conditioned medium was collected
and frozen at ⫺80°C until assayed for apelin release. Apelin
release was determined using the RIA (Phoenix Pharmaceuticals,
Burlingame, CA) as described below.
Radioimmunoassay. An apelin RIA was performed according to the
manufacturer’s instructions (Phoenix Pharmaceuticals, Burlingame,
CA); however, the tissue extraction was modified. For the detection of
total apelin content in the chorionic villi, individual samples from
normal and preeclamptic pregnant women were tested. The RIA assay
recognizes the COOH-terminal peptide and therefore detects all forms
of apelin including apelin-12 and -13; (Pyr1)-13, -17, and -36; and the
prepropeptide-(23–77) and is designated immunoreactive apelin with
direct RIA assessment. The minimum detectable level of the RIA is 4
pg/tube. Tissue homogenization conditions were developed to coincide with the HPLC separation; the homogenization procedure was
different from that recommended for the RIA kit. Specifically, tissues
(100 mg) were homogenized in 10 ml acid ethanol (80% vol/vol 0.1
N HCl) solution containing the peptidase cocktail of inhibitors for
angiotensin peptides except without the rat renin inhibitor (10). The
supernatant of the tissue samples was extracted using C-18 Sep-Pak
columns activated with 5 ml of acid methanol followed by 20 ml of
0.1% heptaflurobutyric acid (HFBA). The sample was applied and the
column was washed with 10 ml 0.1% HFBA. The column was then
washed with 5 ml H20 and then eluted with 3.3 ml of acid methanol
(0.1% HFBA and 80% methanol). The sample was then dried in a
Savant vacuum centrifuge (Farmingdale, NY) and reconstituted for
the RIA (41). Radiolabeled apelin was added to the sample when
homogenized and followed through the extraction process. The recovery of radioactive apelin was between 60 and 65%. The total
apelin concentrations were corrected for recoveries.
HPLC analysis. HPLC and RIA were applied to resolve the
following apelin peptides: apelin-12 and -13; and (Pyr1)-apelin-13,
-17, and -36 using the appropriate standards [apelin-12: Anaspec,
Fremont, CA; apelin-13: Cayman Chemical, Ann Arbor, MI; (Pyr1)apelin-13: Anaspec; apelin-17: Abcam, Cambridge, MA; and apelin36: Anaspec]. The analysis of apelin molecular forms was performed
using placental samples of 200 mg each that were randomly pooled
from either four normal pregnant or four preeclamptic women collected at the time of delivery. The pooled samples were initially
fractionated on a Shimadzu Prominence HPLC with a Nova Pak C18
column (2 ⫻ 150 mm; Waters, Bedford, MA) using 0.1% HFBA (A)
and 80% acetonitrile (B) solvent system at a flow rate of 0.35 ml/min
and a gradient of 30 –50% B over 20 min. Further separation of lower
molecular weight forms of apelin was achieved using a gradient of
32– 40% B over 25 min on the same HPLC system. Fractions were
collected at 1-min intervals, completely evaporated, and analyzed by
the apelin RIA. Products were identified by comparison to apelin
standards. To determine the elution time of oxidized apelin peptides,
1 mg/ml of (Pyr1)-apelin-13 or apelin-12 was incubated with 0.05%
H2O2 for 15 min and then run on HPLC.
Real-time quantitative PCR. Quantitative real-time reverse transcriptase, real-time PCR (qPCR) was performed to assess preproapelin or apelin receptor mRNA concentrations in the chorionic
villi obtained from the normotensive and preeclamptic women as
previously described (57). All probes were purchased from Life
Technologies (Grand Island, NY). 18S ribosomal RNA (rRNA),
amplified using the TaqMan Ribosomal RNA Control Kit (Life
Technologies), served as an internal control. The results were
quantified as Ct values, where Ct is defined as the threshold cycle
of PCR at which amplified product is first detected and expressed
as the ratio of target/control.
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The processing of the apelin precursor is complex and may
yield a number of bioactive peptides. The peptide is synthesized as a 77 amino acid preprohormone that is processed into
a 55 residue intermediate and then to a 36 residue peptide,
apelin-36; further processing of apelin-36 generates apelin-17,
apelin-13, and apelin-12 (5, 31). In addition, the proprotein
convertase PCSK3 or furin can hydrolyze the apelin propeptide
directly to apelin-13 in adipocytes (42). In contrast, angiotensin-converting enzyme 2 (ACE2) acts on the COOH terminus
of apelin-13 and apelin-36 to cleave the Pro-Phe bond with
high catalytic efficiency (42) and constitutes a pathway for
apelin degradation. Additionally, pyroglutamate apelin-13
[(Pyr1)-apelin-13] is a posttranslationally modified form of
apelin-13 containing the pyroglutamate group at the NH2
terminus of the peptide. The 12 COOH-terminal amino acid
peptide is considered the minimal required sequence for apelin
biological activity (40). Presently, it is not known whether
differences exist in the expression of the various molecular
forms of apelin in the human placenta or if the expression
patterns of apelin forms are altered in preeclampsia. In this
study we compared the immunoreactive apelin content in
preeclamptic chorionic villi to villi obtained from women with
normal pregnancy using a quantitative radioimmunoassay
(RIA) that recognizes multiple forms of apelin. Using reversephase high-performance liquid chromatography (HPLC), we
established the expression patterns of the apelin peptides in
normal and preeclamptic villi. In addition, we determined
whether ANG II influences apelin release as one of the mechanisms of apelin regulation in the human chorionic villi.
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E854
APELIN PEPTIDES IN HUMAN PLACENTA IN PREECLAMPSIA
RESULTS
A summary of the patient population characteristics is
shown in Table 1. There were no differences in the biological
age between women with normal and preeclamptic pregnancies. Preeclamptic women had pregnancies that were on the
Table 1. Patient population profile
Characteristics
Norm (n ⫽ 22)
PRE (n ⫽ 20)
Age, yr
Body mass index, kg/m2
Birth weight, g
Gestational age, wk
Systolic blood pressure, mmHg
Diastolic blood pressure, mmHg
Proteinuria (0–4⫹)
23.6 ⫾ 1.1
31.1 ⫾ 1.3
3,218.0 ⫾ 141.6
38.2 ⫾ 0.6
144.2 ⫾ 3.8
80.5 ⫾ 2.3
None
24.3 ⫾ 1.3
36.4 ⫾ 2.1*
2,699.9 ⫾ 177.6*
36.6 ⫾ 0.6*
173.0 ⫾ 3.1*
106.4 ⫾ 1.6*
⬎1⫹
Data are means ⫾ SD. Norm, normal pregnancy; PRE, preeclamptic
pregnancy. *P ⬍ 0.05 vs. normal pregnancy.
average 1.6 wk shorter; their babies were 16% smaller. The
majority of women in the preeclamptic group were diagnosed with a late-onset form of preeclampsia (4). Preeclamptic subjects had 17% higher body mass index compared with normal pregnancy group. Systolic and diastolic
blood pressures were greater in the preeclamptic group vs.
normal pregnancy group (Table 1).
As shown on Fig. 1A, the content of total apelin determined
by direct RIA was significantly lower in preeclamptic chorionic villi, compared with normal villi (n ⫽ 20 –22; P ⬍ 0.05).
Tissue mRNA levels of apelin prepropeptide tended to be
lower in preeclamptic chorionic villi (n ⫽ 15–18; P ⬍ 0.09;
Fig. 1B), although this difference did not attain statistical
significance. However, the protein and mRNA levels of the
APJ receptor were not different between the two groups (Fig.
1, C and D). Figure 1E shows the immunocytochemical distribution of APJ receptor in normal and preeclamptic chorionic
villi: the APJ receptor was identified in syncytiotrophoblasts
depicted by black arrow, cytotrophoblasts by black arrowhead,
and endothelial cells of fetal capillaries by red arrow of human
chorionic villi. The distribution pattern of APJ immunostaining
appeared to be similar in normal and preeclamptic chorionic
villi. Syncytiotrophoblasts are lining the lacuna system. For the
most part, these cells had strong cytoplasmic staining. The fetal
blood vessel endothelium staining was also positive. Villi are
supported by mesenchymal cells that are responsible for producing the intercellular matrix. Both the endothelium (Fig. 1E)
and mesenchymal cells (not shown) were positive for APJ;
however, the staining appears not to be as strong as that
observed in the syncytiotrophoblasts.
The apelin forms in the placenta were then fractionated by
HPLC separation and RIA detection of the resultant fractions. The initial HPLC method resolved apelin-36 and
apelin-17 from the smaller isoforms; however, this gradient
did not distinguish apelin-12, apelin-13, and (Pyr1)-apelin-13 (Fig. 2). The HPLC assessment of pooled samples
from normotensive and preeclamptic tissue revealed that the
smaller isoforms including apelin-12, apelin-13, and (Pyr1)apelin-13 were the primary immunoreactive peaks with little
or no evidence of endogenous apelin-17 and apelin-36 (Fig.
2). To better resolve the smaller apelin forms, a shallower
gradient was employed for the HPLC fractionation that fully
resolved (Pyr1)-apelin-13 and partially resolved apelin-12
and apelin-13; the larger forms (apelin-36 and apelin-17)
were retained to a far greater extent and these fractions were
not collected for RIA measurement (Fig. 3). This second
HPLC analysis revealed that the two major apelin peaks
were (Pyr1)-apelin-13 (14-min elution) and oxidized (Pyr1)apelin-13 (10-min elution) in both preeclamptic and normal
villi (Fig. 3B, top inset). We also noted two immunoreactive
peaks corresponding to apelin-13 (17 min) and apelin-12 (18
min) in pooled tissues from both preeclamptic and normal
chorionic villi (Fig. 3, A–C). Finally, an additional immunoreactive peak that eluted at 15 min appears to be the
oxidized form of apelin-12 and was also evident in both
groups (Fig. 3B, bottom inset).
Since ACE2 may contribute to apelin processing, we utilized
a fluorescent assay to quantify activity in the villi. In Fig. 4 we
show that the preeclamptic chorionic villi exhibit significantly
higher ACE2 activity compared with normal villi (n ⫽ 18 –22;
P ⬍ 0.05).
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Immunohistochemistry. Tissues were fixed in formalin and 70%
ethanol, embedded in paraffin and cut into 5-␮m sections. Immunostaining was performed using the Avidin Biotin Complex (ABC)
method with 0.1% diaminobenzene solution used as the chromogen as
described previously (56). Antigen retrieval treatment with sodium
citrate buffer (pH 6.0) was applied at 90 –95°C for 30 min. Nonspecific binding was blocked in a buffer containing 10% normal goat
serum, 1% Triton-X in PBS for 30 min. Chorionic villi sections were
incubated with the rabbit polyclonal anti-APJ primary antibody (dilution: 1:1,000; Millipore, Merck, Darmstadt, Germany) and secondary biotinylated goat anti-rabbit antibody (dilution: 1:400; Vector
Laboratories, Burlingame, CA). APJ distribution was examined under
a light microscope and photographed with a QImaging Retiga 1300R
CCD digital camera and SimplePCI (Version 6) software.
Western blot hybridization. Placental (100 ␮g of the total protein)
homogenates were prepared for the detection of APJ receptor by
Western blot hybridization as described previously (55). Rabbit affinity purified polyclonal anti-APJ antibody recognizing APJ at 62
kDa (Millipore; Merck) was applied overnight at 1:600 dilution at
4°C. The data were quantified using densitometry (MCID software);
the results were expressed as arbitrary units of intensity and normalized to ␤-actin intensities as described previously (55). Mouse cerebellum tissue lysate was used as a positive control.
ACE2 activity assay. An ACE2 fluorescence assay was performed
according to Vickers et al. (53) with modifications. Reaction mixtures
containing the substrate, 50 mM 7-methoxycoumarin-4-acetyl-alanine-proline-lysine-(2,4dinitrophenyl)-OH, or human placenta, and 10
mM HEPES, pH 7.0, with 1.0 M NaCl were incubated at 37°C for 60
min with inhibitors to block residual ACE, neprilysin, or carboxypeptidase A activity. A second set of reactions contained the selective
ACE2 inhibitor C16, a generous gift of the Millennium, to ensure that
the measured enzyme activity was ACE2. The reaction was terminated by adding 0.2% trifluoroacetic acid; the fluorescence was
quantified in a Perkin Elmer LS 50B fluorometer (excitation: 320 nm;
emission: 405 nm).
Statistics. Comparisons of apelin content, APJ protein, preproapelin, or APJ mRNA levels between the normal pregnancy and preeclamptic groups were performed using unpaired Student’s t-test. The
release of apelin into the media over time (0, 2, and 16 h) was
analyzed using two-way ANOVA followed by the Bonferroni’s posttest (GraphPad Software, San Diego, CA). Two-way ANOVA with
Bonferroni’s posttest was also used to detect differences in apelin
release among the baseline (0 h), ANG II treatment alone or ANG II
plus losartan. A P ⬍ 0.05 was considered statistically significantly
different. All data are presented as means ⫾ SE except the data in
Table 1 that are shown as means ⫾ SD.
APELIN PEPTIDES IN HUMAN PLACENTA IN PREECLAMPSIA
A
B
C
D
E855
Fig. 1. The characterization of apelin content and receptor (APJ) in human chorionic villi obtained from women with preeclampsia vs. women with normal
pregnancy. A: content of immunoreactive apelin in the chorionic villi, *P ⬍ 0.05 vs. normal pregnancy; individual samples of n ⫽ 20 –22. Data are means ⫾
SE. Norm, normal pregnancy; PRE, preeclamptic pregnancy. B: mRNA levels of preproapelin in the chorionic villi. The gene expression data are expressed as
the ratio of apelin to 18S rRNA; P ⬍ 0.09; n ⫽ 15–18. C: protein levels of APJ determined by Western blot analysis in the chorionic villi; n ⫽ 5 in each group.
Representative bands for APJ and ␤-actin are shown. D: mRNA levels of APJ in the chorionic villi; n ⫽ 16 –18. E: immunoreactive distribution of APJ in the
chorionic villi: APJ was identified in syncytiotrophoblasts (black arrow), cytotrophoblasts (black arrowhead), and endothelial cells of fetal capillaries (red arrow).
Magnification: ⫻40.
We then determined the extent that villi may release apelin
peptides using an ex vivo incubation system coupled to apelin
detection by direct RIA. In Fig. 5A, we show a significant
increase in unstimulated release of apelin from chorionic villous explants from women with normal pregnancy at 2 and
16 h and preeclamptic pregnancy at 16 h. As shown on Fig. 5B,
baseline release of apelin at 2 h tended to be lower in the
preeclamptic villi (P ⬍ 0.09); a low dose of ANG II (1 nM)
significantly inhibited the release of apelin only from the
normal villi at 2 h. Addition of the AT1R antagonist losartan
abolished the ANG II-induced decrease in apelin release from
chorionic villous explants. Moreover, losartan enhanced apelin
release above the 2-h baseline levels in both normal and
preeclamptic villi (Fig. 5B).
DISCUSSION
Preeclampsia is a life-threatening disorder specific to pregnancy with no current treatment. The only cure for this disease
is delivery of the placenta, which is considered the primary
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E
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APELIN PEPTIDES IN HUMAN PLACENTA IN PREECLAMPSIA
A
C
Minutes
Fig. 2. HPLC analysis of apelin forms in the human chorionic villi in pooled
samples from women with normal or preeclamptic pregnancy. The pooled
samples were fractionated using 0.1% heptaflurobutyric acid (A) and 80%
acetonitrile (B) solvent system at a flow rate of 0.35 ml/min and gradient
conditions of 30 –50% (B) in 20 min. Data are reported as pg/tube; a pooled
sample from each group was obtained by combining 4 tissue samples from
each group. A: standard profile. B: profile in normal placenta. C: profile in
preeclamptic placenta.
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B
source of inflammatory and vasoconstrictor factors to initiate
widespread endothelial injury and vasoconstriction, upon release into maternal circulation (35). Understanding molecular
factors that lead to preeclampsia could provide a basis for
therapeutic interventions that may reduce the incidence of this
disease and improve maternal and fetal survival, ultimately
leading to improved health of the whole population. The
apelin/APJ system is a novel pleiotropic pathway with a
potential for therapeutic targeting in preeclampsia. However,
the characterization of apelin actions in pregnancy and preeclampsia is complicated by the existence of multiple forms of
the active peptide. To date, the majority of studies on apelin
characterization in human placenta are limited to the analysis
of peptide expression using immunohistochemical methods or
PCR analysis of the precursor protein. However, a complete
analysis of the apelin system in human placenta requires a
greater resolution of the molecular forms of functional apelin
peptides. The use of RIA provides a quantitative analysis of the
total apelin levels, while a combined HPLC-RIA method
allows for the characterization of the expression patterns of
apelin forms in the human placenta.
Several studies have recently investigated the expression of
apelin in human placenta. Inzukua et al. (20) reported that
apelin mRNA and immunohistochemical peptide levels were
lower in placentas from preeclamptic women. Our findings
agree with this study and demonstrate that total apelin content
in the chorionic villi is 30% lower in preeclamptic women as
quantified by direct RIA. Moreover, the expression patterns of
apelin peptides in the normal and preeclamptic chorionic villi
using HPLC-RIA revealed that (Pyr1)-apelin 13, apelin-13,
apelin-12, and possibly oxidized apelin-12 were the major
forms in both normal and preeclamptic chorionic villi. The
presence of the pyroglutamate moiety prevents enzymatic
breakdown by aminopeptidases, thus preserving the biological
activity of the peptide (27). Indeed, this protective group may
contribute to the visibly higher (Pyr1)-apelin-13 peak in chorionic villi of both normal and preeclamptic women relative to
the other molecular forms of apelin. (Pyr1)-apelin-13 was
identified as the predominant form of apelin in the pooled atrial
appendage tissue using HPLC-RIA (36) and in human plasma
by mass spectrometry (58). However, it is important to note
that the current HPLC analysis of apelin isoforms was performed on a small number of pooled samples that precludes a
statistical analysis of the individual peptide isoforms between
groups. Additional studies are necessary to increase the sample
size for the peptide analysis, as well as achieve a more
complete resolution of the apelin forms in the placental tissue.
In addition, a higher body mass index (adiposity) may affect
apelin levels in the preeclamptic women cohort. A higher body
mass index is increasingly associated with an augmented risk
of preeclampsia, and it is one of the contributing factors to the
development of preeclampsia (9, 14). Adiposity leads to the
release of many inflammatory or angiogenic factors, and therefore, it is possible that the biochemical profiles of apelin
peptides are in part influenced by the metabolic activity of
adipocytes (50).
The blood pressure-lowering actions of apelin-12, apelin-13,
and (Pyr1)-apelin-13 are apparent in different animal models
and in patients with chronic heart failure (12, 22, 31, 32, 49).
Intravenous infusion of (Pyr1)-apelin-13 reduced blood pressure and peripheral vascular resistance in heart failure patients
APELIN PEPTIDES IN HUMAN PLACENTA IN PREECLAMPSIA
E857
A
Fig. 3. HPLC analysis of lower molecular
weight apelin forms in pooled samples from
women with normal or preeclamptic pregnancy. Gradient conditions were 32– 40%
(B) over 25 min at 0.35 ml/min (A–C). The
peak at 10 min is consistent with oxidized
form of pyroglutamate apelin-13 [(Pyr1)apelin-13; B, top inset], and the 15-min peak
is consistent with the oxidized form of apelin-12 (B, bottom inset). Products were identified by comparison to apelin standards. A:
standard profile. B: profile in normal placenta with inserts demonstrating the profile
of the oxidized forms. C: profile in preeclamptic placenta. Data are reported as pg/
tube; a pooled sample from each group was
obtained by combining 4 samples from each
group.
C
Minutes
(22). Apelin-12 and apelin-13 reduced both systolic and diastolic blood pressures in anesthetized normotensive Wistar rats
and spontaneously hypertensive rats (31, 32). However, the
magnitude of the acute apelin-13 effect on blood pressure
response was twofold higher than apelin-12, suggesting a
greater potency of apelin-13 (32). Modification of the COOHterminal phenylalanine of apelin-13 inhibited the hypotensive
effect suggesting that phenylalanine is required for the blood
pressure-lowering actions of this peptide (32). Apelin-13 also
decreased blood pressure in a mouse model of atherosclerosis
when administered chronically for 4 wk (12). Therefore, it is
feasible that lower apelin levels in human preeclamptic placenta may diminish its opposing modulation of vasoconstrictor
mediators, which result in an increased blood pressure in
preeclampsia. In addition to effects on blood pressure, (Pyr1)apelin-13 and other apelin peptides may induce vasodilation or
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B
APELIN PEPTIDES IN HUMAN PLACENTA IN PREECLAMPSIA
A
pg/ml
reverse arterial constriction of multiple vascular beds including
mammary, mesentery, coronary, and splanchnic arteries (22,
36). The vasodilatory effects of apelin in vasculature are due to
activation of Akt and endothelial nitric oxide synthase to
generate nitric oxide (12, 21, 49, 59). This is consistent with
studies showing that apelin-induced endothelium-dependent
vasorelaxation is attenuated by coadministration of nitro-Larginine methyl ester, suggesting an endothelium-derived nitric
oxide-dependent mechanism (24). However, apelin may also
induce vasoconstriction via its direct effects on vascular
smooth muscle cells (24, 26, 28, 39). Studies investigating
vascular actions of apelin in pregnancy are necessary to elucidate the role and mechanisms of action of this peptide in
maternal and fetal vasculature.
We noted additional immunoreactive peaks on chromatogram that did not correspond to any of the apelin standard
peptides. Although the exact mechanism for formation of these
additional peaks is presently unknown, increased oxidation
during the tissue-processing protocol may contribute to their
formation. As in our study, an oxidized form of (Pyr1)-apelin-13 was also detected in the heart tissue by HPLC (36) and
this peak eluted at an earlier time point compared with that of
apelin-13 or (Pyr1)-apelin-13.
ACE2, an enzyme of the renin-angiotensin system that
degrades ANG II to ANG-(1–7) and ANG I to angiotensin-(1–
9), is the only known enzyme that can degrade apelin-36 and
apelin-13 with high catalytic efficiency (53). Once phenylalanine on the COOH terminus is released by ACE2 (53), the
peptide may not be recognized by antibodies used for the
apelin RIA. It is possible that ACE2 processes apelin-12 and
apelin-13 peptides to a different extent than (Pyr1)-apelin-13.
Although we did not directly test ACE2 degradation of apelin
forms in human chorionic villi, our data suggest that relatively
lower levels of apelin content in preeclamptic chorionic villi
may reflect higher ACE2 activity.
In regards to the APJ receptor, our data also support other
studies showing the APJ receptor expression in syncytiotrophoblasts, cytotrophoblasts, and endothelial cells of fetal capillaries of human chorionic villi (13, 15), suggesting a paracrine role of apelin in the uteroplacental unit. The angiogenic
properties of apelin as well as apelin/APJ activation of cell
migration and proliferation may be beneficial during the development of the placenta and embryogenesis (23, 30). APJ
B
ANG II
ANG II+LOS
Fig. 5. Time course (A) and the effect of ANG II alone or a combination of
ANG II with losartan (B) on apelin release into conditioned media from
placental chorionic villous explants isolated from women with normal or
preeclamptic pregnancy. The data are expressed as pg apelin/ml; n ⫽ 3–9.
LOS, losartan. *P ⬍ 0.001 vs. Norm 0 h; #P ⬍ 0.0001 vs. PRE 0 h; P̂ ⬍ 0.05
vs. Norm baseline (BL) at 2 h; &P ⬍ 0.001 vs. Norm ANG II; ␥P ⬍ 0.01 vs.
PRE ANG II; ␲P ⬍ 0.05 vs. PRE BL at 2 h.
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Fig. 4. Angiotensin-converting enzyme 2 (ACE2) activity in human chorionic
villi obtained from women with preeclampsia vs. women with normal pregnancy. Data are means ⫾ SE. *P ⬍ 0.05 vs. normal pregnancy; individual
samples of n ⫽ 18 –22.
mRNA or protein expressions were not different between
studied groups in our patient cohorts. Furuya et al. (15)
reported higher APJ mRNA levels in the placentas obtained
from a cohort that included women with gestational hypertension without preeclampsia combined with women with preeclampsia compared with normal pregnancy. Because of their
combined group analysis, it is difficult to compare the results
of their study with ours. In addition, a decreased APJ immunostaining was described in the early onset preeclamptic villi at
the third trimester compared with normal placenta (13, 15).
Plasma apelin measured at delivery was reported to be lower
(6), not different (29), or higher (20) in women with preeclampsia compared with women with normal pregnancy. It is
known that apelin levels are influenced by endogenous enzyme
inhibitors that are present in the plasma (58). We note that
these studies did not attempt to fractionate the various molecular forms of apelin. Aminopeptidases, serine proteases, and, to
a lesser extent, carboxypeptidases are likely responsible for the
processing of apelin peptides to lower molecular weight products or inactive apelin fragments in human plasma (58). Differences in patient population characteristics, the type of assay
used to quantify apelin, and sample collection conditions may
account for the discrepancy of these results. In addition, maternal plasma levels are dependent on placental levels of
apelin, as the removal of half of the fetomaternal unit increases
maternal plasma levels by 23% (51). This observation would
be consistent with degradative enzymes in the placenta that
pg/ml
E858
APELIN PEPTIDES IN HUMAN PLACENTA IN PREECLAMPSIA
GRANTS
This work was supported by the STaRT Pilot Award II from the Division
of Surgical Sciences of Wake Forest School of Medicine (to L. M. Yamaleyeva). We gratefully acknowledge grant support provided in part by Unifi,
Greensboro, NC and the Farley-Hudson Foundation, Jacksonville, NC.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: L.M.Y. and K.B.B. conception and design of research; L.M.Y., L.A., D.L.C., S.S., C.M., and N.T.P. performed experiments;
L.M.Y., M.C.C., D.L.C., N.T.P., and P.E.G. analyzed data; L.M.Y., M.C.C.,
K.B.B., P.E.G., R.T., D.C.M., and H.L.M. interpreted results of experiments;
L.M.Y. prepared figures; L.M.Y. drafted manuscript; L.M.Y., M.C.C., and
K.B.B. edited and revised manuscript; L.M.Y., M.C.C., K.B.B., L.A., D.L.C.,
S.S., C.M., N.T.P., P.E.G., R.T., D.C.M., and H.L.M. approved final version of
manuscript.
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