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* Maternal and Fetal Medicine Section, Institute of Medical Genetics, Yorkhill, Glasgow, G3 8SJ, U.K.;
Department of Obstetrics and Gynecology, University of Cincinnati, College of Medicine, Cincinnati, Ohio, 45267-0526, USA; and
Department of Pathology and
§ Department of Obstetrics and Gynaecology, University of Newcastle upon Tyne, Royal Victoria Infirmary, Newcastle upon Tyne, NE1 4LP, U.K.
1Correspondence: Maternal and Fetal Medicine Section, Institute of Medical Genetics, Yorkhill, Glasgow, G3 8SJ, United Kingdom. E-mail:f.lyall{at}udcf.gla.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: pregnancy • spiral artery • HO-1 • HO-2 • protoporphyrin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Abnormal vascular
development and function is thought to be key in the development of the
pregnancy-specific conditions of pre-eclampsia and intrauterine growth
restriction (2
, 3)
.
Along with the changes in the fetal circulation, the maternal
uteroplacental circulation adapts to pregnancy through striking changes
in the uterine vasculature. During early human pregnancy, extravillous
cytotrophoblast (CTB) from anchoring villi invade the decidualized
endometrium and myometrium (interstitial trophoblast) and also migrate
in a retrograde direction along the spiral arteries (endovascular
trophoblast), transforming them into large diameter conduit vessels of
low resistance (4)
. This physiological transformation is
characterized by a gradual loss of the normal musculoelastic structure
of the arterial wall and replacement by amorphous fibrinoid material in
which trophoblast cells are embedded (5
6
7
8
9
10)
. These
physiological changes are required for a successful pregnancy. Failure
of spiral artery transformation has been well documented in
pre-eclampsia, fetal growth restriction without maternal hypertension
(7
, 11
12
13
14
15)
and miscarriage (11
, 16
17
18)
.
Despite the importance of spiral artery transformation, the mechanisms
that control these processes are poorly understood.
Spiral artery transformation is thought to result from the loss of
normal musculoelastic structure due to CTB invasion. However, vascular
changes have been reported before endovascular CTB invasion has
occurred (4
, 19)
. Pijnenborg et al. (4)
have
related these vascular changes to the presence of interstitial CTB,
suggesting that these cells may produce vasoactive mediators.
Carbon monoxide (CO) is produced by hemeoxygenase (HO), a
microsomal enzyme that oxidatively cleaves heme, a pro-oxidant, to
produce biliverdin and CO in the presence of NADPH-cytochrome
P450 reductase and NADPH (20)
. CO,
like nitric oxide, activates soluble guanylate cyclase to produce cGMP
(21)
. Soluble guanylate cyclase has been purified from the
placenta (22)
and guanylate cyclase activity has been
shown in purified cytotrophoblasts (23)
.
HO consists of two homologous isoenzymes: HO-1, which is inducible, and
HO-2, which is constitutive (24
, 25)
. HO-1 is expressed at
high concentrations in the spleen and liver, where it is responsible
for the destruction of heme from red blood cells. HO-1 can be induced
by numerous stimuli (26
27
28
29
30
31
32
33)
. HO-2 is not thought to be
inducible and is widely distributed throughout the body. CO acts as a
neurotransmitter (33)
, inhibits platelet aggregation
(34)
, and is a vascular smooth muscle relaxant
(35)
. These functions of HO implicate a possible role for
CO in the development and maintenance of maternal and fetoplacental
blood flow, and this hypothesis is tested in the present study.
We hypothesize that HO isoforms will be expressed in the human placenta and placental bed and that inhibitors of HO will increase placental perfusion pressure. Thus, the aims of the study were 1) to determine the expression patterns of HO isoforms within the placenta and placental bed, with an emphasis on trophoblast and vessel endothelial expression, and to determine 2) the functional role of CO in the fetoplacental circulation by studying the effects of infusing an inhibitor of HO, zinc protoporphyrin (ZnPP-9), in the isolated perfused placenta.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies
Cytokeratin (LP34) and mouse smooth muscle actin monoclonal
antibodies were obtained from Novocastra (Newcastle-upon-Tyne, U.K.).
The platelet endothelial cell adhesion molecule (PECAM) antibody was
purchased from R&D Systems (Oxon, U.K.). HO-1 (SPA-895) and HO-2
(OSA-200) rabbit polyclonal antibodies were obtained from Stressgen
Biotechnologies Corp. (Victoria, Canada). The HO-1 antibody was raised
against rat HO-1 purified from a recombinant Escherichia
coli expression system and the HO-2 antibody was raised against
purified native rat testis HO-2. Due to the highly conserved nature of
HO-1 and HO-2 between species, these antibodies can be used to detect
human proteins. There is no cross reaction between the HO-1 and HO-2
antibodies used for both Western blots and immunohistochemistry.
Western blotting
Placental samples were full thickness blocks from chorionic
plate through to basal plate and therefore include stem, intermediate
and terminal villi. Tissue samples (five separate term placentas) were
ground to a fine powder in liquid nitrogen with a mortar and pestle and
added to 4 volumes of cold lysis buffer (25 mM Tris/0.25 M sucrose/1 mM
EDTA, pH 7.6, and 50 µl/g tissue protease inhibitor mixture (Sigma
Chemical Co., Poole, U.K.). Using a Polytron homogenizer at setting 10,
the sample containers were surrounded by ice and homogenized for 3 x 10 s intervals. The homogenate was spun at 5000 x
g for 10 min at 4°C to remove debris, and the resultant
supernatant was spun again at 50,000 x g for 20 min at
4°C to pellet the membranes. The supernatant containing the cytosolic
fraction was aliquoted and stored at -70°C. The membrane pellet was
resuspended in 25 mM Tris (pH 7.6) and spun again at 50,000 x
g for 20 min at 4°C. The supernatant was again removed and
discarded, and the membrane pellet resuspended in 25 mM Tris pH 7.6
(500 µl buffer per gram of starting material) and stored at -70°C.
Protein concentrations of both the cytosol and membrane fractions were
determined by the method of Lowry (36)
, using bovine serum
albumin as a standard, and diluted to the required concentration.
Samples (membrane or cytosol) were mixed 1:1 with loading buffer (1.2
ml 1 M Tris, pH 6.8, 2 ml glycerol, 4 ml 10% sodium dodecyl sulfate, 2
ml 1 M dithiothreitol, 0.8 ml distilled water with bromphenol blue
added to give a deep blue color) and boiled for 5 min before loading.
Samples were separated on 10% sodium dodecyl sulfate-polyacrylamide
resolving gels with a 4% stacking gel using minigel kits (Bio-Rad,
Hemelhempstead, U.K.) (37)
at a constant current of 15 mA.
Each well was loaded with 25 µg of protein. Molecular weight markers
(Sigma, SDS-7B prestained 33–205 kDa range) were loaded beside the
samples.
Protein was transferred overnight in buffer containing 25 mM Tris, 190 mM glycine, and 20% methanol at a constant 30 V to BioBlot NC nitrocellulose membranes (Costar, Corning Inc., Corning, N.Y.). Filters were blocked for 1 h at room temperature in TBSTB buffer (20 mM Tris, pH7.5, 0.5 M NaCl, 0.4% Tween 20, and 0.25% bovine serum albumin) containing 5% normal donkey serum. Both HO-1 and HO-2 antibodies were prepared at a concentration of 1:1000 in TBSTB containing 5% normal human serum and preabsorbed for 1 h at room temperature to reduce nonspecific binding before being used for immunodetection. Omission of this step resulted in many nonspecific bands appearing on the autoradiograph. The antibodies were added for 1 h at room temperature. The filters were rinsed once, washed twice for 5 min in TBSTB, and incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG (SAPU, Carluke, U.K.) diluted 1:2000 in TBSTB for 1 h at room temperature. Blots were then rinsed again and washed twice in TBSTB, followed by one 5 min wash in distilled water. Proteins were detected using the Amersham ECL detection system and filters were exposed to Hyperfilm ECL (Amersham, Buckinghamshire, U.K.).
Immunohistochemistry
Immunohistochemistry was performed using the Vectastain
Universal kit (Vector Laboratories, Peterborough, U.K.). Sections (6
µm) were cut on a cryostat and mounted on glass slides that had been
soaked in acetone for 5 min, 2% silane in acetone for 5 min, washed in
water for 30 min, and then air dried. Each specimen was stained with
hematoxylin and eosin for histological analysis. To assist in
identification of spiral arteries and trophoblast, sections were
immunostained for cytokeratin (1:200) to detect trophoblast, smooth
muscle actin (1:250) to detect muscle, and PECAM (1:500) to detect
endothelium. For HO immunostaining, sections (which were used
immediately after air-drying) were fixed in 1% paraformaldehyde for 5
min, dehydrated in 100% ethanol for 5 min, and rehydrated in water for
2 x 5 min. These and all subsequent steps were performed at room
temperature. Nonspecific binding sites were blocked by incubation with
a blocking agent (Biogenex, San Ramon, Calif.) for 15 min in a
humidified chamber; after washing in TBSTB for 5 min, the sections were
incubated with either HO-1 or HO-2 antibodies for 45 min. Both were
used at a concentration of 1:250 in TBSTB containing 5% normal human
serum and, as for the Western blots, were preabsorbed for 1 h in
this buffer before immunodetection in order to reduce nonspecific
binding. After 2 x 5 min TBSTB washes, the biotinylated secondary
antibody was added for 30 min at room temperature. Two more TBSTB
washes were performed and then endogenous peroxidase activity was
quenched by incubating the sections in 1% (v/v) hydrogen peroxide in
methanol for 15 min. For PECAM, smooth muscle actin and cytokeratin
antibodies acetone fixed sections were blocked with the blocker
supplied with the Universal kit for 30 min at 37°C, washed in
phosphate-buffered saline (PBS) 2 x 5 min, and the primary
antibody (diluted in blocking buffer) was added for 90 min at 37°C.
After 2 x 5 min PBS washes, the secondary antibody was added for
30 min at 37°C. Two more PBS washes were performed and endogenous
peroxidase activity was blocked with 1% hydrogen peroxide in methanol
for 15 min at room temperature. The remaining steps were performed
according to the instructions supplied with the kit. Immunoreactive
proteins were detected with Fast diaminobenzidine tablets (Sigma).
Sections were counterstained in Harris’s hematoxylin (BDH, Poole,
U.K.) and mounted in synthetic resin. Omission of primary antibody or
substitution of nonimmune serum for the primary antibody were included
as controls and resulted in no immunostaining. Intensity of
immunostaining was scored on an arbitrary scale where 0 represents no
staining, 1 represents light staining, 2 represents moderate staining,
and 3 represents dark staining. The scoring of the samples was
performed by two separate observers, both blinded to the tissue
identity. Sections were all stained on the same day to eliminate
day-to-day variations in immunostaining.
Placental perfusions
Placentas were collected immediately after uncomplicated vaginal
delivery or elective caesarean section under a protocol approved by the
University of Cincinnati institutional review board and transported to
the laboratory. Perfusion of placentas was conducted as described
previously (38)
. A suitable third or fourth order
chorionic artery and corresponding vein of an intact cotyledon were
cannulated at a point immediately before passage of vessels through the
chorionic plate to allow perfusion of the fetal circulation. To
establish perfusion of the maternal-placental circulation, the
intervillous space was cannulated with butterfly needles inserted
through the remnants of the spiral arteries in the basal plate. The
effluent from the intervillous space was collected by gravity into a
Plexiglas (Rohm Haas, Philadelphia, Pa.) cone over which the cotyledon
was placed, maternal surface downward. Perfusion medium was Hank’s
balanced salt solution containing 1g/l glucose, 25 g/l
Polyvinylpyrrolidone K30 (Acros, N.J.), 1 g/l bovine serum albumin,
20,000 U/l heparin, and 48 mg/l gentamicin (Fujisawa). The pH of the
medium was adjusted to 7.4 with bicarbonate and gassed with 95% oxygen
5% carbon dioxide at 37°C. Perfusion rates were 4 and 10 ml/min for
the fetal and maternal circulations, respectively. Lateral pressure was
measured in fetal and maternal inflow lines adjacent to the point of
cannulation. Data on pressure, inflow and outflow,
PO2, and pH were sampled every second and stored
on a computer using the Windaq Data Acquisition Software (Dataq
Instruments, Akron, Ohio). Values for O2
consumption are 0.12
µmol·min-1·g-1
tissue. Our figures for glucose consumption 0.67 ± 0.12
µmol·min-1·g-1 and
lactate production 0.67 ± 0.10
µmol·min-1·g-1
agree with other published figures.
Zinc protoporphyrin (ZnPP-9) was used to inhibit HO activity
(39)
. However, since it is known that metalloporphyrins
are (albeit substantially weaker) inhibitors of nitric oxide synthase,
both nitric oxide synthase and prostaglandin activity were inhibited
prior to infusion of the ZnPP-9. In each protocol, once a cotyledon had
been cannulated successfully on both maternal and fetal sides, it was
left to equilibrate for at least 30 min, and the baseline remained
stable after this time. N
-nitro-L-arginine
methyl ester (L-NAME) (1x10-3 M) and
Meclofenamate (1x10-6 M) were then added to the
perfusion medium to block nitric oxide synthase and prostaglandin H
synthase activity, respectively. Perfusions continued for 30 min with
these inhibitors. A stock concentration of 10 mM ZnPP-9 was prepared by
dissolving 31.25 mg in 500 µl pyridine and diluted to 5 ml with
perfusion medium. Since ZnPP-9 is light sensitive, safety illumination
was used when the chemical was exposed. ZnPP-9 was then infused into
the fetal placental circulation using an automated syringe pump at
concentrations of 3.5, 11, 35, 110, and 350 µM to block heme
oxygenase activity. This was performed on five separate placentas. In
control experiments, 10% pyridine in medium was infused in resting
placentas at the same rates as for the ZnPP-9 concentration series;
this was performed on three separate placentas. Each concentration of
ZnPP-9 or vehicle was infused for 5 min, after which the next
concentration followed immediately. Pressure values used in the data
herein are maximum values, 3 min after infusion of each concentration
of ZnPP-9 or vehicle. The integrity of the system was determined at the
beginning and end of experiments by ascertaining constrictor responses
to U46619 and dilator responses to GTN. All data were normalized to
remove the influence of resting pressure and weight. Data were not
normalized to placental weight since not only does this assume that the
vascular volume bears a constant relationship to cotyledonary weight,
which is not true, but normalizing for placental weight actually adds
greater variance to the data (40)
.
Statistical analysis
Where appropriate, statistical comparisons for
immunohistochemistry were performed using the Kruskal-Wallis 1-way
analysis of variance test to determine whether there was a significant
difference between the three groups for endothelial or trophoblast
immunostaining. Comparison between paired groups was then performed
using the Mann-Whitney U test. For perfusion studies,
statistical analysis was performed using the Kruskal-Wallis 1-way
analysis of variance test, followed by the Mann-Whitney U
test for comparisons at each concentration of ZnPP-9. Statistical
differences were taken to be significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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depicts Western blot analysis of both the membrane and cytosolic
fraction of third trimester placenta preparations and shows that HO-1
(Fig. 1
, upper panel) was undetectable in both the membrane (M) and
cytosolic fraction (C). In contrast, HO-2 protein (Fig. 1
, lower panel)
was located in the membrane fraction (M). The size of the HO-2 was
estimated from molecular weight markers at 36 kDa, which agrees with
the size reported for other human tissues. Four representative samples
are shown. Western blots were not performed throughout gestation
because the subsequent immunohistochemical results showing temporal and
spatial changes in expression within different cell types would have
made the results uninterpretable. Thus, the Western blot data showed
that HO-2 was present in term placenta, and was a microsomal enzyme
with a molecular weight similar to that reported by others. No
differences in HO-1 or HO-2 expression were found in blocks taken from
different areas within a placenta, and so these data are not shown.
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Immunohistochemistry: placentas
Thirteen first trimester (8–13 wk gestation), nine second
trimester (14–19 wk gestation) and eight third trimester placentas
(28–40 wk gestation) were studied. The data for HO-2 immunostaining
are shown in Table 1
. Syncytiotrophoblast and cytotrophoblast immunostaining was similar in
all samples. In the first trimester, dark HO-2 immunostaining was seen
primarily in the trophoblast layer and only occasional light staining
was noted on endothelial cells. Significant differences were noted in
trophoblast immunostaining between the three groups
(P<0.0005): staining was reduced in third trimester samples
compared with both first (P<0.0005) and second trimester
(P<0.0005) samples. Representative immunostains are shown
in Fig. 2
. Endothelial immunostaining was also different in the three groups
(P<0.00001): staining was greater in second compared to
first trimester samples (P<0.05) and in third compared to
second trimester samples (P<0.0005).
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Extravillous trophohoblast of cell islands were cytokeratin positive
(Fig. 2C
), and these cells were also positive for HO-2 (Fig. 2D
). Trophoblast cells that had broken through the
syncytiotrophoblast and formed cell columns were all positive for HO-2
(Fig. 2E
). Similarly, cytokeratin-positive extravillous
trophohoblasts (EVT) in the cytotrophoblastic shell identified by
cytokeratin-positive staining (Fig. 2F
, left panel) were
also HO-2 positive (Fig. 2F
, right panel). There were no
differences in intensity of immunostaining in EVT throughout gestation.
Within the placenta, the immunostaining pattern for HO-1 was different
from HO-2. Most samples showed negligible HO-1 staining on villous
tissue throughout gestation, although occasionally parts of the
syncytiotrophoblast layer were positive. There were no differences in
staining throughout gestation. Figure 3A
shows a placenta at 10 wk gestation and Fig. 3B
shows a placenta at term. An interesting feature was the intense HO-1
immunostaining on syncytial sprouts (Fig. 3A
). In contrast
to the situation with villous tissue, EVT HO-1 immunostaining was
comparable to that of HO-2 throughout gestation. Figure 3C
shows a cell island at 16 wk gestation depicting EVT immunostained with
cytokertatin. A matched section immunostained with HO-1 (Fig. 3D
) shows that these cells are also HO-1 positive. An
example of the staining pattern of HO-1 on cell columns is shown on a
16 wk sample in Fig. 3E
. A striking observation on many of
the cell columns was the lack of HO-1 immunostaining in the first few
cell layers of the column whereas the more distal cells, including
those that had invaded the decidua, were HO-1 positive.
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At term, EVT in the cytotrophoblastic shell, identified by cytokeratin
immunostaining (Fig. 3F
, left panel), were also HO-1
positive (Fig. 3F
, right panel). There were no
differences in intensity of immunostaining in EVT throughout gestation.
Our immunohistochemical studies revealed no differences in expression between the different anatomical sites of the placenta.
Immunohistochemistry: placental bed
Twenty six placental bed biopsies were examined: 12 from
pregnancies evenly spread between 8 and 13 wk gestation and 14 from
pregnancies evenly spread between 14 and 19 wk gestation. Nine
placental bed biopsies from term pregnancies were also included for
comparison.
Histological examination of specimens showed the presence of interstitial CTB in both decidua and myometrium in all specimens between 8 and 13 wk gestation. The number of CTBs in the myometrium increased with gestational age.
HO-2 was expressed in both intravascular and interstitial trophoblast
throughout gestation. Figure 4A, B
shows a section containing a spiral artery within
decidua at 8 wk gestation. Cytokeratin immunostaining of this section
shows the presence of both interstitial and intravascular trophoblasts
(Fig. 4A
). A matched section immunostained with HO-2 shows
that both interstitial and intravascular trophoblasts are also HO-2
positive (Fig. 4B
). Other noncytokeratin-positive decidual
cells present throughout the first trimester were HO-2 positive.
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Histologial examination of specimens from 14 to 18 wk gestation
revealed extensive interstitial CTB invasion within both decidua and
myometrium. Intravascular invasion was apparent in myometrial as well
as decidual vessels. Figure 4C, D
shows a myometrial vessel
at 16 wk gestation that is surrounded by trophoblasts. Trophoblasts
were identified by cytokeratin immunostaining (Fig. 4C
), and
these same cells were HO-2 positive (Fig. 4D)
. Immunostaining, albeit
much less intense, was also noted on myometrium.
While trophoblast invasion is complete by the third trimester, EVT in
third trimester placental bed biopsies remained HO-2 positive. This is
illustrated in Fig. 4E, F
. Figure 4E
shows a
biopsy containing several areas with vessels surrounded by
trophoblasts. These cells were HO-2 positive (Fig. 4F
).
The immunostaining pattern for HO-1 within the placental bed was the
same as for HO-2, with all extravillous interstitial, endovascular, and
perivascular trophoblast being HO-1 positive. An example is shown in
Fig. 4G, H
. In Fig. 4G
a myometrial vessel is
surrounded by a trophoblast that has been immunostained with
cytokeratin. A matched section shows that these cells are also HO-1
positive (Fig. 4H
).
Perfusion studies
The effect of infusion of ZnPP-9 on fetoplacental perfusion
pressure is shown in Fig. 5
. Infusion of ZnPP-9 led to a dose-dependent constriction. Infusion of
vehicle alone had no effect on perfusion pressure: At 3.5 µM ZnPP-9,
the mean percent baseline pressure was 102.4 ± 2.2 and
100.67 ± 2.0 in the treatment and control groups, respectively,
and at 11 µM the values were 99.8 ± 1.6 and 98.67 ± 0.88
in treatment and control groups, respectively. At 35 µM, the value of
the treatment group was 106 ± 1.5, which was significantly higher
than the control group 95.33 ± 2.3 (P<0.05). At 110
µM ZnPP-9, the percent baseline pressure increased to 126.4 ±
9.9 compared with the control group, which was 91 ± 4.4
(P<0.05). At the highest concentration infused, 350 µM
ZnPP-9, the percent baseline pressure had increased further to
212.2 ± 30; this was also significantly higher than the vehicle
control, which was 86.7 ± 6.1 (P<0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CO binds to heme and activates soluble guanylate cyclase
(21)
. NO, produced by the enzyme nitric oxide synthase
(NOS), also binds to the heme prosthetic moiety of the soluble
guanylate cyclase, leading to increased cGMP production and smooth
muscle relaxation. Discrepancies in localization of NOS and guanylate
cyclase in the brain indicate that a substantial portion of guanylate
cyclase may not serve as a target for NO (33)
. It has been
proposed that HO-2 and eNOS may have complementary and co-coordinated
physiological roles (39)
.
Human placental syncytiotrophoblast express the endothelial type nitric
oxide synthase (eNOS) (41
42
43
44)
. NO produced by
syncytiotrophoblast has at least three physiological targets: the
intervillous space, autocrine effects on trophoblast function, and
paracrine interactions with villous core components. Nitric oxide
inhibits platelet aggregation and leukocyte adhesion while modulating
the immune response. We speculate that, in keeping with the
complementary roles of HO-2 and NOS, HO-2 on syncytiotrophoblast may
have overlapping roles with those proposed for eNOS in
syncytiotrophoblast. Similar effects by CO produced by
syncytiotrophoblast would benefit both maternal blood flow through the
intervillous space and avoidance of immune recognition of the
fetoplacental allograft. We found that HO-2 expression on
syncytiotrophoblast was significantly higher in early pregnancy
compared with the third trimester. One explanation is that higher
amounts of CO may be required in early pregnancy to establish blood
flow from the spiral arteries to the intervillous space. However, once
blood flow is established and spiral artery transformation is complete,
the need for CO production would be reduced.
In contrast to the data with HO-2, HO-1 protein was expressed at low to undetectable levels in most areas of the placental villous tree throughout gestation. In a small number of villi, occasional small areas of syncytiotrophoblast expressed HO-1. The very low amounts of HO-1 in placenta were confirmed by the Western blotting experiments, which, in contrast to the situation with HO-2, did not reveal HO-1 protein in any of the samples studied. EVT did, however, express HO-1. An interesting finding was the positive HO-1 immunostaining noted on syncytial sprouts within the intervillous space. Whether HO-1 is expressed on these cells after they are shed from the villi or whether HO-1 expression is up-regulated during this process remains to be established.
In an earlier study (45)
we showed that EVT do not express
eNOS or iNOS, suggesting that NO is not involved in spiral artery
dilatation. In contrast, in the present study we found that HO-2 was
expressed on EVT of cell columns, cell islands, and invading
interstitial CTB. These findings suggest that CTB-derived CO may
contribute to dilatation of the spiral arteries during early pregnancy
and may also be involved in the invasion process itself.
We also found in this study that that HO-2 immunostaining was present
in myometrium, but this does not reflect nonspecific staining. We had
previously performed a comprehensive study of HO expression in human
myometrium and shown by using Western blotting, immunohistochemistry,
and reverse transcriptase-polymerase chain reaction that HO-2 is
expressed by myometrium (46)
. With regard to stromal
staining, many more cells were positive in the first trimester when
compared with term (Fig. 2A, B
). This temporal pattern
further confirms the specificity of the immunostaining for these cells.
eNOS is also expressed on villous endothelial cells, and NO produced
from these cells is believed to be an important vasodilator within the
placental vasculature (47
48
49
50
51)
. In this study we found
that HO-2 expression was significantly higher in villous endothelial
cells in third trimester placentas when compared with first and second
trimester samples. It may be that as the pregnancy advances, increased
HO-2 expression on endothelial cells would result in more CO production
and facilitate blood flow as the demands by the growing fetus are
increased.
The antibody NCL-LP34 reacts with cytokeratins 5, 6, and 18
intermediate filament proteins in frozen tissues. Cytokeratins 8, 18,
and 19 have been shown to be expressed by all villous and extravillous
subsets of CTB throughout human pregnancy (52)
Numerous
studies have used antibodies against cytokeratins 8 and 18 to detect
villous and extravillous trophoblast. Although this antibody stains
glandular epithelial cells as well as villous and extravillous
trophoblasts, the characteristic morphological appearance of glands
means that this staining does not confound interpretation. However,
unlike other antibodies directed against cytokeratin 8/18, LP34 does
not react with myometrial cells, particularly in frozen sections. Thus,
LP34 was ideal for identifying trophoblast in this study.
The human placenta lacks autonomic innervation, and thus blood flow is
regulated by humoral agents or autocrine/paracrine mechanisms
(1)
. Vasodilators such as bradykinin, acetylcholine,
histamine, and the calcium ionophore A23187, which increase nitric
oxide release in other vascular systems, do not appear to have
vasodilator effects in the perfused placenta; however, nitric oxide
does contribute to the maintenance of basal tone and attenuates the
actions of vasoconstrictors in the perfused placenta
(47
48
49
50
51)
. We have shown here that the inhibitor of HO,
ZnPP-9, increased resistance in the perfused placenta in a
dose-dependent manner, suggesting that CO also contributes to the
maintenance of basal tone in the placenta. Metalloporphyrins have been
reported to have substantially weaker effects on nitric oxide synthase
activity (39)
and on prostaglandin release
(53)
. To eliminate confounding the effects of these two
pathways, NO and prostaglandin release was blocked, prior to infusion
of ZNPP-9, with L-NAME and Meclofenamate, respectively.
ZnPP-9 produced a dose-dependent constriction within the placental
vasculature, implying a functional vasodilatory role for CO within this
circulation. The doses used in these studies were based on previous
in vitro (54)
and in vivo studies
(55)
. Metalloporphyrins have been used extensively as HO
inhibitors to study the functional role of CO in a number of tissues.
However, there is evidence that metalloporphyrins may affect cGMP
levels independent of HO activity. ZnPP-9 has been reported to inhibit
(56)
and stimulate (57)
NOS activity. To
remove any possible confounding effect on NO production, we inhibited
NO synthase prior to infusion of ZnPP-9. Serfass and Burstyn
(58)
have recently suggested that ZnPP-9 may directly
affect soluble guanylate cyclase in vitro. The authors
reported stimulation of guanylate cyclase with concentrations of ZnPP-9
between 25 and 250 nM and inhibition at higher concentrations. However,
the authors also reported that the heme-loading status of the guanylate
cyclase plays a role in the effect observed with ZnPP-9 making
extrapolations to the in vivo state complex. Previous animal
studies have reported inhibition of guanylate cyclase with ZnPP-9
concentrations of 100 µM and greater (39)
. Thus,
although direct inhibition of soluble guanylate cyclase may be a
confounding factor at high concentrations of ZnPP-9, it is unlikely to
completely account for the constrictor response observed in the present
study.
The expression and functional role of hemeoxygenases has not been
investigated in the human placenta. Odrcich et al. (59)
have studied HO expression in the guinea pig placenta and HO-1
expression in a single term human placenta. Although this study
reported results different from our data, studies of guinea pig may
have limited relevance to the human situation (45)
and
cell columns do not exist in rodents. One other explanation for the
differences in the guinea pig study compared with this study of human
placenta may be due to differences in methodology. We found that
without preabsorption of the HO antibodies to eliminate nonspecific
binding, nonspecific bands appeared on Western blots and nonspecific
immunostaining occurred on both cryosections and paraffin sections of
placenta. In the study by Odrcich, the antibodies were not preabsorbed,
nor were Western blots performed to check the specificities of the
antibodies.
In summary, understanding the control of trophoblast invasion is an
area of great physiological importance, with potential implications for
failed pregnancy. In pre-eclampsia, there is failure of normal
transformation of the myometrial spiral arteries (14)
.
Less is known about fetal growth retardation in the absence of maternal
hypertension. Uteroplacental blood flow is reduced (60)
,
and there is increasing evidence that this is due to defective
placentation. Several studies have shown that there are morphological
abnormalities in uterine spiral arteries in some cases of isolated
fetal growth retardation (9
, 12
, 61
, 62)
. Failure of
spiral artery transformation may also be an important cause of
miscarriage (11
, 17
, 18
, 63
, 64)
. This study suggests a
role for both isoforms of HO in trophoblast invasion and spiral artery
transformation, and functional studies suggest that CO may operate as a
vasodilator in the fetoplacental circulation. Further studies,
including those on failed pregnancy, are warranted.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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