J Appl Physiol
90: 2420–2426, 2001.
Cotyledon and binucleate cell nitric oxide synthase
expression in an ovine model of fetal growth restriction
HENRY L. GALAN,1 TIMOTHY R. H. REGNAULT,2 TIMOTHY D. LE CRAS,2,3
R. WESLIE TYSON,4 RUSSELL V. ANTHONY,2 RANDALL B. WILKENING,2
AND STEVEN H. ABMAN2,3
Division of Perinatal Medicine in the Departments of 1Obstetrics and Gynecology and 2Pediatrics;
3
Pediatric Heart Lung Center, University of Colorado Health Sciences Center; and 4Department of
Pathology, Childrens Hospital, Denver, Colorado 80262
Received 22 February 2000; accepted in final form 31 January 2001
INTRAUTERINE GROWTH RESTRICTION (IUGR) is a significant
complication of pregnancy affecting up to 8% of all
pregnancies (6, 28). Fetal complications due to IUGR
include intrapartum distress, hypoxia, asphyxia, and
intrauterine demise. Neonatal complications include
meconium aspiration, metabolic and hematologic disturbances, cognitive dysfunction, and cerebral palsy.
In addition to perinatal morbidity and mortality, epi-
demiological studies suggest that IUGR may contribute to adverse health effects in adulthood including
cardiovascular disease and diabetes (4, 27). IUGR fetuses may exhibit symmetric or asymmetric patterns of
growth. Although the etiologies of asymmetric IUGR
are heterogeneous, the clinical and pathological manifestations may be due to universal structural and functional abnormalities in the placenta.
A number of placental structural abnormalities have
been described in IUGR pregnancies, including decreased villous number, diameter, and surface area, as
well as decreased arterial number, lumen size, and
branching (10, 14, 18, 21, 31). Doppler ultrasound
studies of the umbilical artery in both humans and
sheep provide evidence for an increase in placental
vascular resistance in IUGR pregnancies (9, 38). Increased vascular tone and reactivity may be due, in
part, to alterations in vasoactive mediators such as
endothelin, prostacyclin, thromboxane, and nitric oxide (NO). Hence, increased placental vascular resistance may reduce umbilical blood flow and nutrient
delivery to the fetus.
NO is a free radical molecule with potent vasodilator
properties and a short half-life that is synthesized by
the enzyme NO synthase (NOS). NOS catalyzes the
conversion of L-arginine to L-citrulline with NO as a
by-product. NO activates soluble guanylate cyclase to
produce cGMP that results in vasorelaxation (23).
Three isoforms of NOS have been described: endothelial NOS (eNOS), inducible NOS, and neuronal NOS.
Gestational age-related changes in eNOS have been
described in the umbilical artery and in a number of
fetal tissues (23). eNOS has been localized to syncytiotrophoblast and vascular endothelium in humans
and to the vascular endothelium in sheep, baboon, rat,
and guinea pig (45). NO is a vasoactive molecule that
has been shown to be active in a number of vascular
beds including the brain, lung, and kidney (1, 24, 36,
39) as well as the placenta and umbilical vessels (41,
45). Past studies have shown that chronic treatment
Address for reprint requests and other correspondence: H. L.
Galan, Dept. of Obstetrics and Gynecology, Univ. of Colorado Health
Sciences Center, 4200 E. 9th Ave., Campus Box B-198, Denver, CO
80262.
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
endothelial nitric oxide synthase; hyperthermia; placenta;
binucleate cell; intrauterine growth restriction
2420
8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society
http://www.jap.org
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Galan, Henry L., Timothy R. H. Regnault, Timothy D.
Le Cras, R. Weslie Tyson, Russell V. Anthony, Randall
B. Wilkening, and Steven H. Abman. Cotyledon and binucleate cell nitric oxide synthase expression in an ovine model
of fetal growth restriction. J Appl Physiol 90: 2420–2426,
2001.—Heat exposure early in ovine pregnancy results in
placental insufficiency and intrauterine growth restriction
(PI-IUGR). We hypothesized that heat exposure in this model
disrupts placental structure and reduces placental endothelial nitric oxide synthase (eNOS) protein expression. We
measured eNOS protein content and performed immunohistochemistry for eNOS in placentas from thermoneutral (TN)
and hyperthermic (HT) animals killed at midgestation (90
days). Placental histomorphometry was compared between
groups. Compared with the TN controls, the HT group
showed reduced delivery weights (457 ⫾ 49 vs. 631 ⫾ 21 g;
P ⬍ 0.05) and a trend for reduced placentome weights (288 ⫾
61 vs. 554 ⫾ 122 g; P ⫽ 0.09). Cotyledon eNOS protein
content was reduced by 50% in the HT group (P ⬍ 0.03).
eNOS localized similarly to the vascular endothelium and
binucleated cells (BNCs) within the trophoblast of both experimental groups. HT cotyledons showed a reduction in the
ratio of fetal to maternal stromal tissue (1.36 ⫾ 0.36 vs.
3.59 ⫾ 1.2; Pⱕ 0.03). We conclude that eNOS protein expression is reduced in this model of PI-IUGR and that eNOS
localizes to both vascular endothelium and the BNC. We
speculate that disruption of normal vascular development
and BNC eNOS production and function leads to abnormal
placental vascular tone and blood flow in this model of PIIUGR.
PLACENTAL ENOS EXPRESSION IN AN OVINE MODEL OF IUGR
MATERIALS AND METHODS
Experimental design and animal care. This study was
approved by the University of Colorado Health Sciences
Center Animal Care and Use Committee. Eight mixed-breed
(Columbia-Rambouillet) ewes with time-dated singleton
pregnancies were used for this study. Beginning at 40 days
gestation (term ⫽ 147 days), four ewes were placed in a HT
environment and four ewes were kept at ambient conditions
serving as TN controls. The environmental chamber conditions have been previously described and include a temperature that cycles at 40°C for 12 h during the day and 35°C
during the night and humidity that is kept between 30 and
40% (9). All ewes were pair fed and offered water ad libitum.
Sheep were euthanized at 85–93 days gestation, and fetal
and placentome weights were recorded. The placentome was
divided into cotyledon (fetal) and caruncle (maternal) components, which were frozen in liquid nitrogen. Placentomes
were also sectioned and placed in 10% formalin and paraffin
embedded for histology and immunolocalization studies.
Western blot analysis. Caruncles and cotyledons were homogenized separately in buffer containing leupeptin, phenylmethylsulfonyl fluoride, betamercaptoethanol, and pepstatin
A. Western blot analysis was performed on crude homogenates of whole caruncles and cotyledons from each animal (25
␮g of total protein) by use of a monoclonal antibody to eNOS
(Transduction Laboratories, Lexington, KY), as previously
described (16). Western blots were performed with NuPAGE
4–12% Bis-Tris gradient gels (Novex, San Diego, CA). Densitometry was performed with a UMAX PowerLook II scanner (UMAX DATASystems, Hsinchu, Taiwan) and National
Institutes of Health Image software. Western blot analysis
was used to compare eNOS protein content between 1) caruncles and cotyledons from TN pregnancies, 2) cotyledons of
increasing weights from TN pregnancies, and 3) cotyledons of
comparable weights from HT and TN pregnancies (3.86 ⫾ 0.2
vs. 3.72 ⫾ 0.1 g, respectively; P ⫽ 0.63)
Histology and immunohistochemistry for eNOS protein.
Midline cross sections of placentomes from each treatment
group were fixed in 10% buffered formalin and embedded in
paraffin. Paraffin sections 6 ␮m thick were mounted onto
Superfrost Plus slides (Fisher Scientific, Fairlawn, NJ) for
staining with hematoxylin and eosin and for immunohistochemistry.
A single pathologist (R. W. Tyson) who was blind to the
treatment groups performed the histomorphometry on hematoxylin- and eosin-stained sections. A single placentome of
comparable size from each animal was analyzed. Vessel density and vessel diameters were measured for surface vessels
of the placentome. Vessel density was performed by point
counts using a grid eyepiece and standard magnification
(⫻100). Counts were taken when cross points of the grid
touched the wall of the fetal or maternal vessel or fell within
the lumen. Using an eyepiece micrometer, we then measured
the vessel wall thickness and the vessel outside diameter for
vessel wall thickness-to-diameter ratios. This ratio was calculated by a formula previously described by Abman et al. (2)
for pulmonary arterioles. The area of fetal villi and maternal
stroma were measured on three random ⫻10 microscopic
fields, and the ratio of fetal villi to maternal stroma was
compared between treatment groups by using Image-Pro
Plus morphometric image analysis software (Media Cybernetics, Silver Spring, MD). Images were captured by a Polaroid digital microscopic camera. Once the images were
captured, the areas of fetal tissue were outlined and the total
fetal tissue area was calculated. The total area of the image
was calculated, and the fetal area was subtracted to give the
maternal tissue area. The ratio of fetal to maternal area was
then calculated. Figure 1 is an example of the computercaptured image of a ⫻4 histological view of a sheep cotyledon
demonstrating the acquisition of the areas of interest outlined in black.
Immunohistochemistry for eNOS was performed on 6-␮mthick sections of paraffin-embedded whole placentomes.
Slides were dewaxed with 100% xylene. Slide preparation
and antigen retrieval were performed as previously described
by Le Cras et al (16). Slides were washed in PBS, and
sections were blocked for 45 min with Super Block (Sky Tek,
Logan, Utah) diluted 1:10 (vol/vol) in 1⫻ PBS. Slides were
then incubated for 2 h with a rabbit polyclonal primary
antibody against eNOS (Santa Cruz Biotechnology, Santa
Cruz, CA) at a dilution of 1:2,000 or with an IgG1-negative
control (Jackson Laboratories, West Grove, PA). Sections
were then washed in 1⫻ PBS. Sections were then incubated
Fig. 1. Example of the Image Pro Plus morphometric image demonstrating how fetal (F) and maternal (M) tissues are outlined. Areas
(mm2) are reported on a separate display screen (not shown).
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with an antagonist that inhibits all NOS isoforms
causes IUGR and preeclampsia-like syndrome in the
pregnant rat (22, 44). However, information on the role
of placental NOS and its role in the etiology of IUGR is
limited.
We hypothesized that heat-stress exposure early in
ovine pregnancy disrupts placental development and
reduces placental eNOS protein expression. To address
this hypothesis, we used an established ovine model of
placental insufficiency and intrauterine growth restriction (PI-IUGR) induced by hyperthermia (HT) beginning early in gestation. This model is characterized by
asymmetric fetal growth and reduced placental mass
(8). It is also associated with reduced uterine and
umbilical blood flows, reduced transplacental amino
acid flux, reduced glucose and oxygen transport capacity, as well as abnormal umbilical arterial and aortic
Doppler velocimetry (3, 5, 9, 30, 37). To test our hypothesis, eNOS protein expression of placentomes from
90-day-gestation HT animals and thermoneutral (TN)
control sheep were compared by Western blot analysis,
immunohistochemistry, and histology.
2421
2422
PLACENTAL ENOS EXPRESSION IN AN OVINE MODEL OF IUGR
Table 1. Gestational age and fetal and placental
weights for each study group
Age at delivery, days
Fetal weight, g
Placental weight, g
TN
HT
P value
93.5 ⫾ 1.2
631 ⫾ 21
554 ⫾ 122
93.3 ⫾ 1.4
457 ⫾ 49
288 ⫾ 61
0.89
0.05
0.09
Values are means ⫾ SE. TN, thermoneutral group; HT, hyperthermic group.
RESULTS
Table 1 shows gestational ages and fetal and placental weights at death for each study group. Both groups
were delivered at similar gestational ages. Fetal
weight was significantly reduced in the HT group (P ⬍
0.05). A trend for a decrease in placental weight was
noted in the HT group.
Figure 2 shows a representative Western blot for
eNOS in cotyledon and caruncle pairs for the TN
group. These findings show that eNOS protein is predominantly located in the cotyledon component of the
placentome. The cotyledon has 14-fold higher eNOS
protein concentration compared with caruncle. Figure
Fig. 2. Representative Western blot demonstrating a 14-fold greater
endothelial nitric oxide synthase (eNOS) protein concentration in the
cotyledons (n ⫽ 4) compared with caruncles (n ⫽ 4) from an animal
in the thermoneutral (TN) group (P ⬍ 0.001).
Fig. 3. Representative Western blot showing a positive correlation
between eNOS protein concentration and cotyledon mass (R2 ⫽
0.84).
3 depicts a representative Western blot and corresponding linear regression plot of cotyledons of different weights (25 ␮g protein load for each) from TN
controls. Linear regression analysis shows a strong
correlation in TN controls (r2 ⫽ 0.8) between eNOS
protein and cotyledon size. Figure 4 shows a representative Western blot with corresponding histogram of
the densitometry demonstrating a twofold decrease in
eNOS protein content in the cotyledons of the HT
pregnancies compared with the TN group (P ⬍ 0.05).
Figure 5 shows immunolocalization of eNOS protein to
chorionic binucleate cells within the fetal trophoblast
and to the endothelium of the blood vessels within the
fetal villi.
Histomorphometry showed that there were no differences between HT and TN groups for either fetal surface vessel counts or wall thickness (Table 2). However,
there was a reduction in the ratio of fetal to maternal
stroma in the HT group compared with TN controls
(P ⬍ 0.03). Figure 6 shows the striking histological
differences in fetal to maternal stromal content between the HT and TN groups. The histology of the HT
cotyledons was similar to that of a term cotyledon that
is shown for comparison.
Fig. 4. Representative Western blot depicting a 2-fold reduction in
the cotyledons from the animals in the hyperthermic (HT) group (n ⫽
4) compared with TN controls (n ⫽ 4; P ⬍ 0.05).
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for 45 min with a biotin-labeled anti-mouse (for IgG1) or
anti-rabbit (for eNOS) secondary antibody at a dilution of
1:10,000. Slides were washed in 1⫻ PBS. Slides were incubated in streptavidin-biotin-horseradish peroxidase solution
and developed with diaminobenzidine (DAB) using the Vectastain ABC and DAB kit (Vector Laboratories, Burlingame,
CA). The NiCl enhancement-DAB color development reaction
was stopped by washing with water. Slides were dehydrated
in 70% ethanol-30% water, 95% ethanol-5% water, 100%
ethanol, and 100% xylene and then coverslipped.
Statistical analysis. Gestational age, cotyledon number,
fetal and placental weights, and Western blot densitometry
values between treatment groups were compared by Wilcoxon test by use of SigmaStat 2.0 (SPSS, Chicago, IL).
Histomorphometry between HT and NT groups was compared by unpaired t-tests. Data are presented as means ⫾
SE, and a P value of ⬍0.05 was considered significant.
PLACENTAL ENOS EXPRESSION IN AN OVINE MODEL OF IUGR
2423
Fig. 5. A and B: localization of eNOS to
the binucleated cells in the trophectoderm at ⫻40 and ⫻100 magnification,
respectively. C: IgG negative control.
D: eNOS localization to the vascular
endothelium within the fetal villi.
We found that HT animals showed a twofold reduction in cotyledon eNOS protein content. We also found
in control animals that eNOS protein content was
14-fold greater in the cotyledon compared with the
caruncle. The strong positive correlation of eNOS protein with cotyledon weight is important from a methodological standpoint. This finding directed us to compare cotyledons of similar sizes between the TN and
HT groups, thus avoiding the potential bias of comparing different-sized cotyledons. Alternatively, by comparing cotyledons of similar sizes, we may have
masked larger differences in eNOS protein content
because the HT animals tended to have placentas of
smaller weight. Previously shown to localize to vascular endothelium, we found that eNOS protein also
localized specifically to the BNCs in the trophectoderm
of the cotyledon. No differences in fetal surface vessel
number or wall thickness were detected; however,
there was a 2.5-fold decrease in the ratio of fetal villous
stroma to maternal villous stroma in the HT cotyledons
relative to TN cotyledons. This decrease in the fetal
villous stroma to maternal villous stroma in the HT
cotyledon was strikingly similar to that normally seen
at term. This suggests an accelerated maturation of the
placenta in the HT animals, a finding that has been
previously described in the human IUGR placenta (11).
As in previous studies using this model of IUGR, we
found a significant decrease in fetal weight in the HT
Table 2. Placentome histomorphometry
NT
Surface vessel counts
Surface vessel wall
thickness
Fetal-maternal stromal ratio
Values are means ⫾ SE.
HT
P value
11.5 ⫾ 2.5
10.3 ⫾ 3.6
0.78
0.54 ⫾ 0.03
3.59 ⫾ 1.2
0.56 ⫾ 0.03
1.36 ⫾ 0.36
0.53
0.03
group. Interestingly, this decrease in fetal weight was
not accompanied by a significant reduction in placental
weight. However, it has been previously shown that
placental weight in the near-term ovine fetus is dependent on the duration of heat stress (9). Thus it is likely
that, with time, differences in placental weight would
have become apparent.
A reduction in eNOS protein in the HT cotyledons
provides a potential explanation for the decrease in
absolute umbilical blood flow (37, 30) and increase in
placental resistance detected by umbilical artery Doppler (9) previously shown in this PI-IUGR model. Reduced eNOS protein would likely result in reduced NO
production and increased basal vascular tone and vascular resistance.
Past studies have provided evidence supporting a
role for NO in regulating tone in the fetoplacental
circulation in early gestation. The NO-cGMP cascade
appears to be most active in the first trimester human
placenta. Peak NOS activity in human placental homogenates occurs in the first trimester and decreases
to term (29). Izumi et al. (12) further showed in human
umbilical artery smooth muscle bath studies that the
amount of NO and the sensitivity of the smooth muscle
to NO decreased with advancing gestation. NOS activity has been shown to rise early in the tissues of a
variety of organs in fetal guinea pigs (40). These investigators suggested that NOS activity levels may be
related to estrogen levels because the increased NOS
activity mirrored the rapid rise and plateau of estrogen. Using Doppler ultrasound, Lees et al. (19) showed
that umbilical artery vascular impedance was highest
in early pregnancy, which was paradoxical to the
higher levels of NOS activity and cGMP levels. It may
also be that levels of vasoconstrictors may be high,
which may offset the NO-cGMP cascade. Lees et al.
speculated that perhaps shear stress in these highresistance vascular beds was responsible for the in-
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DISCUSSION
2424
PLACENTAL ENOS EXPRESSION IN AN OVINE MODEL OF IUGR
Fig. 6. A and B: histological sections of
cotyledons from 90-day postconception
TN and HT animals, respectively. Note
the marked disparity in the F to M
components between the groups. C:
histological section of a term TN cotyledon for comparison. Striking similarities are seen between the HT and the
term TN sections.
placenta, BNCs are analogous to invasive cytotrophoblasts in the human placenta. The human cytotrophoblast differentiates to become cell columns that invade
the uterine epithelium to anchor the fetus and establish blood flow to the placenta and fetus (46). The
human cytotrophoblast also produces the syncytiotrophoblast that produces proteins and steroid hormones
(46). Similarly, the BNC produces protein and steroid
hormones. The BNC and the human syncytiotrophoblast also share eNOS localization (45). Given this
information, we speculate that the BNC may be the
ovine placental correlate to the cyto- and syncytiotrophoblast of the human placenta because it has both
hormonal and migratory functions. We further speculate that the BNC develops a vascular phenotype much
like the human cytotrophoblast, which has been shown
to express receptors and other markers of endothelial
cells (46). The localization of eNOS to the BNC in our
study is consistent with this speculation and rational
from a teleological viewpoint.
Because the NO-cGMP cascade is most active in
early gestation, this may provide some insight into the
mechanisms of reduced placental weight and IUGR in
this model. The effects of heat treatment on placental
growth early in gestation may be critical because this
is a period of rapid vascular growth in the placenta.
Although we found no difference in the number or wall
thickness of the surface vessels of the placentomes, it
remains necessary to quantitate vasculature changes
within the fetal villi. Preliminary data on fetal villous
vascular casts from our lab suggest a significant disturbance in angiogenesis of HT placentas (T. R. H.
Regnault, R. V. Anthony, and R. B. Wilkening, unpublished data). Disruption of placental growth and probably vascular growth might result in altered of eNOS
protein expression. The reduction in eNOS protein may
be due to the direct effect of heat on endothelial cells
and BNC or to an overall reduction in the number of
endothelial cells and BNC in proportion to other cells.
Regardless of the mechanism, a reduction in eNOS will
probably affect the NO-cGMP cascade and alter basal
vascular tone and possibly vessel growth. NOS activity
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crease of NOS activity and increased cGMP levels.
Chronic hypoxia has been shown to upregulate eNOS
in the rat lung, and although eNOS levels were high,
Sato et al. (33) showed that NO production was low
under hypoxic conditions. It is possible that NOS activity is higher in early pregnancy because of the relative hypoxic environment of the villi noted in early
pregnancy.
The localization of eNOS protein to the BNC is of
particular interest because it is a trophoblast cell that
also produces progesterone and ovine placental lactogen. The BNC migrates from the fetal side of the
trophectoderm to the maternal syncytium to deliver
these hormones into the maternal bloodstream. In a
study comparing eNOS and inducible NOS isoforms in
the placenta of various species, Zarlingo et al. (45)
found that eNOS localized to both syncytiotrophoblasts
and vascular endothelium in the human placenta, but
only to the vascular endothelium of sheep and other
species. They studied these animals late in gestation,
and it is possible that eNOS expression was undetectable since NOS levels are decreased in late gestation
(29). In our study, immunohistochemistry was performed on placentomes at midgestation, which is a
time in gestation when eNOS expression is higher (29).
In all ruminant placentas, the BNC is found in the
trophectodermal epithelium and are found in fairly
constant proportions across gestation (13, 42, 43). The
BNC has been shown to migrate across the fetomaternal junction (43). BNCs appear to have two primary
functions: they form the maternal-fetal syncytium
needed for implantation and placentomal growth, and
they produce and deliver protein and steroid hormones
(43).
The localization of eNOS to the BNC together with
other known features of the BNC allows us to draw
comparisons with the trophoblast of the human placenta. The BNC is the only migratory cell in the ovine
placenta moving across the fetal-maternal interface to
the maternal uterine epithelium forming the fetalmaternal syncytium necessary for implantation. Because they are the only migratory cells in the ovine
PLACENTAL ENOS EXPRESSION IN AN OVINE MODEL OF IUGR
NOS gene and protein expression in small resistance
vessels of the lung were induced with chronic hypoxia
(17), and inhibition of NO production induces vasoconstriction in rat lungs (26). In contrast, Fike et al. (7)
have shown that chronic hypoxia decreases both NO
production and eNOS synthase in newborn piglet
lungs. In addition, NO appears to play an active role in
regulating basal pulmonary vascular tone as depicted
by an increase in pulmonary vascular resistance with
NOS inhibition (35). Because hypoxia can affect eNOS
and NO production, it remains necessary to determine
the oxygen tensions at midgestation in our IUGR
model so that the mechanism of reduced eNOS protein
can be better defined.
In summary, heat stress reduces fetal and placental
growth, disrupts normal placental histology, and reduces the eNOS protein expression in the cotyledon.
We speculate that the placental vascular development
will mirror the abnormal villous histology and that
this, together with the reduced eNOS production, accounts for the abnormal hemodynamics seen in this
model. It remains uncertain whether the reduction in
eNOS protein is a result of abnormal vascular development or whether a reduction in eNOS protein contributes to abnormal vascular development. The localization of eNOS to the BNC introduces another
variable that could be affected by heat and could impact placental morphology and vascular development.
The availability of an animal model that produces an
asymmetric pattern of growth restriction by the application of a known environmental stress provides a
means of investigating the timing and duration of the
environmental stress in terms of fetal and placental
growth. It also allows for the investigation of pathophysiological and molecular mechanisms of placental
damage induced by thermal stress.
We appreciate the insight of Dr. Frederick C. Battaglia and the
technical support provided by Neil Markham, David Hood, Peter
Orchard, Karen Trembler, Pat Lenhardt, Sarah Williams, and Kate
Fasth.
This work was supported by the American Association of Obstetricians and Gynecologists Foundation and Burroughs-Wellcome
Fund, the National Institutes of Health NHLBI SCOR Grant HL57144, National Institutes of Health Program Project Grant
PO1HD20761, and March of Dimes Grant no. 6-F497-0174.
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and cGMP levels shown to be highest early in human
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Our finding of reduced placental eNOS concentration
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circulation of IUGR fetuses (20, 25). Lyall et al. (20)
determined NO concentrations by measuring indexes
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umbilical venous blood from IUGR and normal pregnancies. They found that pregnancies complicated by
IUGR had significantly higher nitrite and nitrate levels in the umbilical venous plasma. They concluded
that the increase in NO indexes could represent a
compensatory effort to improve placental blood flow.
Myatt et al. (25) used a semiquantitative immunohistochemical technique to show that pregnancies complicated by IUGR or by both preeclampsia and IUGR have
increased basal distribution of eNOS in syncytiotrophoblast as well as intense terminal villous capillary
endothelial immunostaining. Although several possibilities exist to explain the differences between these
studies, one likely explanation is the difference in relative gestational age at the time of tissue collection. If
our samples had been collected later in gestation, as
were the earlier reports (20, 25), we may have seen
comparable increases in eNOS production. In support
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PI-IUGR (37). Greater placental hypoxia could lead to
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production (15). Hypoxia alone has been shown to increase eNOS production in other vascular beds as well.
Thus alterations in eNOS expression in IUGR placenta
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multiple time points during gestation. Therefore, our
findings could still be consistent with those of Lyall et
al. and Myatt et al. The different findings between our
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our model of IUGR.
Although the heat stress IUGR animals have been
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this study is that we did not know the oxygen tension
of these fetuses at midgestation. This may be important because, although little is known about the effects
of hypoxia on eNOS regulation in the placenta, there
are reports suggesting that hypoxia affects eNOS expression in the pulmonary vascular bed. Endothelial
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PLACENTAL ENOS EXPRESSION IN AN OVINE MODEL OF IUGR
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