Department of Pediatrics, University of Texas Southwestern Medical
Center at Dallas, Dallas, Texas 75390
Pregnancy is associated with
increases in cardiac output and uterine blood flow (UBF) and a fall in
systemic vascular resistance. In ovine pregnancy, UBF rises from ~3%
of cardiac output to ~25% at term gestation, reflecting a >30-fold
rise in UBF by term. This increase in UBF supports exponential fetal
growth during the last trimester and maintains fetal well-being by
providing excess oxygen and nutrient delivery. These hemodynamic
changes are associated with numerous hormonal changes, including
increases in placental steroid hormones and enhanced activation of the
renin-angiotensin and sympathetic nervous systems, all of which are
believed to modulate systemic and uterine vascular adaptation and
vascular reactivity. Systemic pressor responses to infused ANG II are
attenuated in normotensive pregnancies and the uteroplacental
vasculature is even less sensitive, suggesting development of
mechanisms to maintain basal UBF and permit the rise in UBF necessary
for fetal growth and well-being. The effects of ANG II on the
uteroplacental vasculature are reviewed, and the mechanisms that may
account for attenuated vascular sensitivity are examined, including ANG II metabolism, vascular production of antagonists, ANG II-receptor subtype expression, and the role of indirect mechanisms.
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INTRODUCTION |
PREGNANCY IS A UNIQUE physiological
state that is responsible for successful propagation of mammalian
species. Normal mammalian pregnancy is associated with numerous
hemodynamic changes, e.g., in sheep, cardiac output increases >50%,
systemic vascular resistance (SVR) falls, arterial blood pressure
decreases modestly, and cardiac output is redistributed (36, 91,
114, 116). Unique to pregnancy is a rise in uterine blood flow
(UBF), which in sheep is >30-fold and reflects an increase from
3-5% of cardiac output in the nonpregnant state to 20-25%
at term gestation (36, 108, 113, 114, 116, 124). This
exponential rise in UBF predominantly occurs in the last two-thirds of
gestation. It is associated with placentation, i.e.,
angiogenesis, followed by vasodilation and is essential for normal
fetal growth and well-being, providing the oxygen and nutrient delivery
required for the exponential fetal growth that parallels the rise in
UBF in the last trimester (113, 116). As UBF increases in
pregnancy it is redistributed within the uterus, and at term >85% of
total UBF is directed toward the maternal placental vascular bed
(87, 115). Thus the rise in UBF is predominantly due to
exponential increases in placental blood flow (108, 124). These cardiovascular changes are associated with modifications in the
endocrine milieu, representing placental- and nonplacental-derived substances, including steroid hormones and products of the
renin-angiotensin system (RAS). Although there is an apparent
interdigitation between the hemodynamic and endocrine alterations
occurring in pregnancy, the precise relationships remain unclear.
In addition to the general cardiovascular modifications described,
there are changes in vascular responsiveness or sensitivity to several
vasoconstrictors. For example, pregnant women develop attenuated
pressor responses to infused ANG II and
-adrenergic agents (1,
20, 49). These changes occur early in pregnancy and are of
interest because the refractoriness to ANG II is lost as early as the
midtrimester in women who later develop pregnancy-induced hypertension
(49, 141). It has been proposed that by understanding this
particular aspect of pregnancy one may subsequently determine the
pathogenesis of this hypertensive disorder, which affects 5-10%
of pregnant women (36). The uterine vascular bed also is
refractory to ANG II in normotensive pregnancies (44, 95, 125), and, similar to the systemic vasculature, this is lost in
the presence of hypertension (45), which is often
paralleled by a fall in UBF, as well as uterine oxygen and nutrient
delivery, and compromised fetal growth and well-being. Thus it would be important to determine what adaptive mechanisms modulate the systemic and uterine vascular beds during normal pregnancy and if these mechanisms are similarly affected in the presence of hypertensive disease.
The changes in the hormonal milieu in normotensive pregnancies are
extensive, and, although they have been reasonably well characterized,
their purposes are far from understood. It is believed that ovarian-
and placental-derived estrogens and progesterone participate in
modifying vascular reactivity and may facilitate the widespread
vasodilation observed in pregnancy (116). The increase in
the activity of the RAS in pregnancy has received a great deal of
attention. It is associated with increases in the synthesis of renin,
angiotensinogen of hepatic and nonhepatic origins, and ANG II,
resulting in increases in plasma renin activity and circulating levels
of ANG II (77, 132, 138). The purpose for these changes
also remains unclear, but may be related to the mechanisms responsible
for modulating the extensive vasorelaxation associated with
normotensive pregnancy. That is, they may participate in maintaining
vascular tone (109) and the rise in cardiac output. Evidence for this is indirectly obtained from studies in which nonspecific blockade of the ANG II receptor (ATR) and/or the
-adrenergic receptor after estrogen exposure in nonpregnant sheep
resulted in substantial falls in mean arterial pressure (MAP; 38).
However, this remains to be proven in pregnancy. It also is unclear why the placental unit has the capacity to synthesize many components of
the RAS (14, 58, 137). Maybe they participate in local modulation of uterine vascular tone or angiogenesis early in gestation. Although this too remains unanswered, it may be essential to our understanding of the systemic and uterine hemodynamic changes in pregnancy.
In the present review, I will examine several aspects of the
relationship between ANG II, the primary vasoactive agent produced by
the RAS, and the uteroplacental circulation. Because much of our
knowledge regarding these interactions has been derived from extensive
animal studies, in large part from the chronically instrumented ovine
species, I will use these data to describe our present state of
knowledge, but I will refer to the human when reasonable correlates are
available, thereby demonstrating the similarities that exist between
the species.
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ANG II AND VASCULAR REACTIVITY: SYSTEMIC VS. UTERINE |
Normal pregnancy is associated with attenuated pressor responses
to systemic infusions of ANG II (1, 20, 49, 141). This is
detected as early as the midtrimester and is no longer evident after
parturition (1). Several species develop a similar refractoriness during pregnancy (9, 11, 104, 120). In
studies from our laboratories we (120) not only
observed attenuated pressor responses to continuous systemic infusions
of ANG II that were evident early in ovine gestation, but also that the
dose-response curves generated from steady-state responses in
nonpregnant and pregnant ewes were strikingly similar to those
published for nonpregnant and pregnant women (141).
Furthermore, the pressor dose, i.e., the dose required to elicit a
20-mmHg rise in MAP, in nonpregnant and pregnant ewes was the same as
that reported for women (141). We (81) later
observed that pregnant ewes, similar to pregnant women
(20), also develop refractoriness to the pressor effects of systemic infusions of
-agonists. These observations, therefore, support the value of using chronically instrumented ewes as a model in
which to investigate the mechanisms responsible for the adaptive
changes in the cardiovascular system associated with normal pregnancy
and may permit us to subsequently develop better strategies to
investigate and understand hypertensive diseases in pregnant women.
In our initial studies of the systemic responses to ANG II in
pregnancy, we implanted electromagnetic flow probes on both main
uterine arteries to continuously monitor UBF, having previously demonstrated the reliability of this method (124). We
observed a dose-dependent response in UBF, but little or no change
occurred until the systemic dose of ANG II exceeded 0.08 µg · kg
1 · min
1 (Fig.
1), which results in pharmacologic plasma
levels of ANG II (93). Furthermore, there was a biphasic
response in UBF during systemic ANG II infusions (Fig.
2). That is, during ANG II infusions >0.1 µg · min
1 · kg
1
there was a rise in UBF that occurred after the more rapid increase in
MAP, followed by a progressive fall in UBF although MAP remained stable, achieving a steady state by 3-6 min. In earlier studies using systemic bolus doses of ANG II and/or anesthetized animals, ANG
II was considered a uteroplacental vasodilator, because UBF rose soon
after ANG II infusions started (5, 48, 69, 89, 134, 142).
These studies, however, 1) frequently used bolus doses of
the peptide, 2) were often limited to examining the initial phase of the UBF response, 3) did not always consider the
simultaneous changes in perfusion pressure, and 4) were
frequently performed in acute animal preparations, which modifies
vascular responses to several agents (116, 123). When we
(95) examined the change in uterine vascular resistance
(UVR) in chronically instrumented animals across a broad range of
continuous systemic ANG II doses and only used the steady-state
responses, there were only increases in UVR at all doses studied,
demonstrating that ANG II was always a uterine vasoconstrictor in
pregnant sheep over a broad range of doses (Fig.
3). This was consistent with observations
by Cohen et al. (28) in anesthetized rabbits and is now
generally accepted (26, 151). On further inspection of the
uterine responses to systemic ANG II infusions, we also observed that
the rise in UVR was significantly less than the rise in SVR at all
doses of ANG II <2.3 µg/min (Fig. 3), suggesting that at
physiological and even pharmacologic doses (93) the
uterine vascular bed was even "less sensitive" to the
vasoconstricting effects of ANG II than the systemic vasculature as a
whole.

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Fig. 1.
Effects of continuous systemic infusions of ANG II (AII) on
uteroplacental blood flow in pregnant ewes between 99 and 143 days of
gestation. The line representing the polynomial regression is
presented. [Reprinted from Rosenfeld and Gant (120) with
permission.]
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Fig. 2.
A
continuous recording of mean arterial pressure and uterine blood flow
(UBF) in a pregnant ewe at 111 days of gestation. [Reprinted from
Rosenfeld and Gant (120) with permission.]
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Fig. 3.
Comparison of the simultaneous relative changes in
systemic and uterine vascular resistance during the continuous systemic
infusion of 5 doses of ANG II in pregnant sheep. Means ± SE are
presented. [Reprinted from Naden and Rosenfeld (95) with
permission.]
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To understand the mechanisms responsible for the biphasic UBF response
to ANG II, we (95) examined the simultaneous relative changes (percent change, %
) in perfusion pressure or MAP, UVR and
UBF. Because each hemodynamic parameter has a different unit of
measurement, the raw data cannot be easily compared; but by controlling
for baseline values and calculating the percent change, a comparison of
the relative responses is obtained, and any interaction between the
three variables is easily assessed (95). When this simple
rearrangement was performed and the data analyzed using dose and
duration of ANG II infusion in the steady state, it became apparent why
others had concluded that ANG II was a uterine vasodilator. At doses of
ANG II
1.15 µg/min, which generally results in physiological plasma
concentrations (93), there was a rise in MAP that always exceeded the rise in UVR and was associated with an increase or no
change in UBF (Fig. 4). However, when the
dose of ANG II exceeded 2.3 µg/min, resulting in high pharmacological
plasma levels (93), the relative rise in UVR exceeded the
increase in MAP and UBF fell. Thus changes in UBF are highly dependent
on the difference in the relative responses in MAP or perfusion
pressure and local UVR (55, 70, 148). A similar
relationship has been observed in studies of UBF and ANG II in pregnant
dogs and guinea pigs and confirmed in the ewe (26, 37,
151). When the responses to individual ANG II doses are analyzed
across time, a strikingly similar relationship is seen. That is, at
"all" doses of ANG II studied, the change in MAP occurs more
"rapidly" than the rise in UVR; thus UBF always increases in the
initial response to systemic ANG II infusions (Fig.
5). However, if the dose of ANG II
ultimately increases UVR greater than MAP in the steady-state response,
e.g., 11.5 µg/min (Fig. 5), UBF clearly falls at this time. Until
recently, we and others did not appreciate the meaning of this
difference in the timing of the systemic and uterine responses to
systemic ANG II infusions. This will be addressed later. Nonetheless,
the conclusions from these studies are 1) ANG II is only a
uterine vasoconstrictor in all species studied under unstressed
conditions as reported for other vascular beds, 2) the
uterine vasculature is less sensitive to systemic ANG II infusions than
the systemic vasculature at physiological plasma levels of the peptide,
and 3) UBF responses in pregnancy must be assessed with
consideration to simultaneous alterations in perfusion pressure. In
other words, UBF is "protected" from the vasoconstrictor effects of
elevated circulating ANG II that occurs during pregnancy (77,
132, 138). With the advent of pulse-gated Doppler for measuring
UBF or resistance in women, Erkkola et al. (44) observed
similar differences in uterine and systemic sensitivity to infused ANG
II in normotensive pregnant women. They later reported that this
uterine refractoriness, as with systemic refractoriness, was lost in
women who developed hypertensive disorders (45). These
data, therefore, provide additional evidence that the ewe is an
excellent model in which to study normal cardiovascular adaptation in
pregnancy and, in particular, the changes in uteroplacental adaptation.
Furthermore, although the placental morphology differs between women
and sheep (116), changes in uterine vascular reactivity
appear to be similar.

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Fig. 4.
Comparison of the relative changes in mean arterial
pressure ( ), uteroplacental blood flow
( ), and UVR ( ), expressed as the
percent of control values, across a range of continuous systemic
infusions of ANG II. Means ± SE are presented. [Reprinted from
Naden and Rosenfeld (95) with permission.]
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Fig. 5.
The simultaneous changes across time in UBF
(A), mean arterial pressure (B), and UVR
(C) during continuous systemic infusions of ANG II. Data for
3 doses are presented; each point represents the mean of 7 experiments.
[Reprinted from Naden and Rosenfeld (95) with
permission.]
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Thus far, the data provide insight into changes in total UBF and UVR.
The pregnant uterus, however, is comprised of at least three tissues:
the myometrium, which makes up the bulk of uterine weight, the
endometrium, and the placental cotyledons, the site of gas and nutrient
exchange. In nonpregnant ewes these tissues (caruncles in the
nonpregnant state are the subsequent sites of implantation and
represent the cotyledons) receive a similar proportion of UBF, ~33%
(123, 124). However, UBF is gradually redistributed during
pregnancy, and the placental portion of UBF increases
disproportionately, accounting for
85% of total UBF by term
(87, 108, 115). Because this portion of UBF is responsible
for fetal growth and well-being, it is important to know if ANG II
alters placental blood flow. To accomplish this, studies were performed
in conscious pregnant ewes using radionuclide-labeled microspheres,
which permit the simultaneous measurements of cardiac output and its
distribution, SVR, the responses of the three uterine tissues, and
responses by nonuterine tissues at specific time points (114,
124, 125). Flow probes were also implanted on each uterine
artery to continuously monitor UBF and more accurately time the
microsphere infusions. As anticipated from earlier studies, the
relative rise in UVR during systemic ANG II infusions <1 µg/min was
less than the rise in SVR, confirming the uterine refractoriness
previously noted with flow probe measurements (95). At ANG
II doses >5 µg/min, the %
UVR exceeded %
SVR and MAP, thus UBF
fell. Importantly, the lower ANG II dose results in estimated plasma
levels of the peptide of ~200 pg/ml (93), which resemble
that observed in normotensive pregnant women (77). The
higher doses, however, result in plasma levels of ANG II >2,000 pg/ml,
which are nonphysiological. Placental blood flow and vascular
resistance were unchanged with 0.573 and 5.73 µg ANG II/min (Fig.
6), and blood flow fell only 16% with
the highest dose studied, 11.5 µg/min. In contrast, endometrial and
myometrial blood flows significantly decreased and vascular resistance
rose with all three doses (Fig. 6). Thus the placental circulation is
refractory to a wide range of systemic ANG II doses. When the
distribution of UBF was calculated, the proportion going to the
placental cotyledons actually rose from 74% before ANG II infusion to
90% with the pharmacologic dose of the peptide, further demonstrating
the protection afforded maternal placental blood flow through increases
in perfusion pressure and decreases in blood flow to the other uterine
tissues.

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Fig. 6.
Dose-dependent effects of continuous systemic infusions
of ANG II on total uteroplacental vascular resistance and vascular
resistance in the individual uterine tissues, i.e., endometrium,
myometrium, and placenta, in near-term pregnant sheep. Blood flows were
measured with radionuclide-labeled microspheres. SE are noted.
*P < 0.05, P < 0.02. [Reprinted
with permission (125)].
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It had been suggested that the differences in the responses in total
SVR and UVR during systemic ANG II infusions might be due to a greater
sensitivity of the nonreproductive compared with reproductive tissues
(95). However, data to support this were lacking. This was
addressed in the microsphere studies when nearly all of the
nonreproductive tissues, including the kidney, adrenal, and adipose,
were observed to consistently decrease blood flow and increase vascular
resistance at all three doses of ANG II studied (Fig.
7). This provided conclusive evidence of
the differences in vascular sensitivity to ANG II that exist between
various tissues/organs in normal gestation. Of interest, myometrial and
endometrial sensitivities resembled that in peripheral or
nonreproductive tissues. Curran-Everett et al. (37)
reported similar differences between uteroplacental and
nonuteroplacental responses to ANG II in late-gestation guinea pigs.
These differences in vascular reactivity are intriguing, because they
suggest that ANG II may be a safer pressor agent for treating
hypotensive episodes in gravid women than the standard use of
-agonists. Support for this is obtained from observations in women
and sheep that ANG II has greater effects on SVR and MAP than UVR,
thereby having minimal effects on UBF. Moreover, all
-agonists
studied have greater effects on UVR than SVR (53, 56, 81, 99,
110, 117, 126), which may be accentuated in pregnant women with
hypertensive disease, but has not been studied.

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Fig. 7.
Dose-dependent effects of continuous systemic infusions
of ANG II on vascular resistance in kidneys, adrenal glands, and
adipose tissue in near-term pregnant sheep. Blood flows were measured
with radionuclide-labeled microspheres. SE are presented.
*P < 0.05, P < 0.02, **P < 0.001. [Reprinted with permission
(125).]
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Although existing data supported the thesis that the uterine
vasculature in pregnancy was less responsive to systemic ANG II
infusions than the systemic vasculature, it was unclear if this was an
inherent characteristic of the uterine vascular bed or due to some
adaptive change(s) that occurs in pregnancy. Cox et al.
(34) addressed this by comparing systemic and uterine responses to systemic ANG II infusions in pregnant and nonpregnant ewes
with flow probes on both main uterine arteries. MAP rose dose
dependently in both groups, but as expected, the %
MAP in nonpregnant ewes greatly exceeded that in pregnant animals at "all"
doses, values increasing 40-65% in the former vs. 20-50% in
pregnant ewes, P < 0.0001. Although UVR rose dose
dependently in both groups, the responses in nonpregnant ewes were
substantially greater at all doses studied (Fig.
8), values rising 80-350% vs. 25-90% in pregnant ewes (P < 0.0001). Thus UBF
fell only 20% in pregnant ewes with the highest ANG II dose studied
compared with 60% in nonpregnant ewes (Fig.
9). When the simultaneous relative changes in systemic and uterine responses were compared, pregnant ewes
had greater relative increases in MAP than UVR at physiological doses,
confirming prior observations (95, 125). In contrast, nonpregnant animals had greater increases in UVR than MAP at all ANG II
doses, resembling responses to
-agonists (81); e.g., with an estimated plasma ANG II level of 0.8 ng/ml the %
UVR was ~130% vs. ~50% for MAP. These data (34), therefore,
demonstrate that the uterine vasculature "develops" the
pregnancy-associated attenuation in ANG II-induced vasoconstriction,
which also occurs with
-agonists (81). Furthermore,
they also suggest that the differences in systemic and uterine
responses to systemic ANG II infusions in normal pregnancy are not
inherent to the uterine vasculature but are due to pregnancy-related
changes. This could reflect the growth and development of the less
responsive placental vasculature, which accounts for >85% of UBF
(125). Alternatively, it might be due to modifications in
ANG II metabolism, synthesis of local ANG II antagonists, changes in
uterine vascular smooth muscle, alterations in ANG II receptor (ATR)
expression and/or binding, or any combination of factors.

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Fig. 8.
Comparison of the effects of comparable systemic and
local intra-arterial infusions of ANG II on the relative changes in UVR
in pregnant (A) and nonpregnant (B) sheep.
Different letters within each group (i.e., systemic and intra-arterial)
denote significant differences in responses across concentrations using
repeated-measures ANOVA, P < 0.001. [Reprinted with
permission (34).]
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Fig. 9.
Comparison of the effects of comparable systemic and
local intra-arterial infusions of ANG II on the relative changes in UBF
in pregnant (A) and nonpregnant (B) sheep.
Different letters within each group (i.e., systemic and intra-arterial)
denote significant differences in responses across concentrations using
repeated-measures ANOVA, P < 0.001. [Reprinted with
permission (34).]
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ANG II AND VASCULAR REACTIVITY: METABOLISM OF ANG II |
One potential explanation for the attenuated systemic pressor
responses to infused ANG II in pregnancy is that the peptide is cleared
from the circulation at a greater rate than that observed in the
nonpregnant state. This also would explain the attenuated uterine
vascular sensitivity associated with pregnancy, but not the difference
between systemic and uterine sensitivity. It had been suggested that
ANG II metabolism was greater in pregnancy and due to increases in
circulating levels of placentally derived aminopeptidases (103,
140) or through enhanced maternal placental clearance
(92), reflecting placental aminopeptidases that also prevent transplacental transport of ANG II to and from the fetal compartment (6, 51, 64, 75, 107). To address this, Naden et al. (93) measured the metabolic clearance of ANG II
(MCRANG II) in nonpregnant and near-term pregnant ewes.
There was the anticipated difference in pressor responses to systemic
ANG II infusions, but over a range of ANG II doses there was no
difference in the MCRANG II, 56.2 ± 6.3 and
55.9 ± 4.3 ml · min
1 · kg
1,
respectively. They also reported that plasma levels of ANG II achieved
during steady-state infusions were proportional to the infusion rate in
both groups and proposed this would permit investigators to calculate
the estimated arterial ANG II concentrations during studies of the
effects of systemic steady-state infusions. The values for
MCRANG II in nonpregnant ewes are consistent with earlier
observations in nonpregnant sheep (46, 47) and the human
(40, 65, 100). Magness et al. (77)
subsequently reported that the MCRANG II was not
significantly different in normotensive nonpregnant and pregnant women,
85 ± 10 vs. 68 ± 3 ml · min
1 · kg
1,
respectively. Moreover, these values resemble those observed in the
ewe, demonstrating another similarity between species (93, 121). The half-life for ANG II was ~49 s, which also is
consistent with other species and women (40, 42). When the
removal rate of ANG II by circulating aminopeptidases was
examined, the estimated half-life was ~10 min (47),
which is inconsistent with removal rates seen in vivo. Thus increases
in circulating aminopeptidase enzymes associated with pregnancy
probably play a minor role in ANG II removal. It also was determined
that the increased volume of distribution in pregnancy could not
account for the attenuated responses.
Subsequently, Rosenfeld et al. (121) examined ANG II
clearance across the uteroplacental vascular bed of pregnant sheep. They confirmed the observations of Naden et al. (93) for
maternal MCRANG II in pregnant sheep and reported that
uteroplacental clearance averaged 20 ± 6% in term animals. This
is in sharp contrast to a fetal MCRANG II of 680 ml · min
1 · kg
1 and >90%
clearance of ANG II across the fetal placental vascular bed (121,
135). If the adult rat kidney clears ANG II at 1 µg · min
1 · g kidney
1
(73) and ovine kidneys weigh ~180 g, renal clearance
could account for removal of a predominant portion of ANG II from the maternal circulation, which is consistent with the data for
uteroplacental clearance. Therefore, in women and sheep neither the
attenuated systemic nor uterine responses to infused ANG II in
pregnancy reflect enhanced ANG II removal from the circulation. More
recently, Iyer et al. (62) reported that neither ATR
subtype is directly involved in ANG II clearance in hypertensive adult
male rats. However, the type 2 ATR (AT2R) appeared to
enhance ANG II clearance. Although the role of the ATR subtypes in
MCRANG II has not been examined in pregnancy, the
AT2R subtype predominates in the uterine vascular bed of
women and sheep (see below; 32, 35), and its role in uteroplacental ANG
II clearance has not been examined.
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ANG II AND VASCULAR REACTIVITY: LOCAL ANTAGONISTS |
Pregnancy is associated with enhanced vascular production of
several vasoactive substances, including PGs (52, 78, 82), estrogens (36, 116), and nitric oxide (NO; 133), which may increase organ and tissue blood flows and antagonize local vascular responses to ANG II or other vasoconstrictors so that blood flow is
maintained. The existing literature on PGs in pregnancy is immense and
cannot be completely examined in this review; therefore, only pertinent
points relative to ANG II and UBF will be addressed.
During pregnancy there are increases in circulating PGs, particularly
vasodilating PGs (52, 82), and in the uterine synthesis of
these compounds (78, 142, 149). Furthermore, treatment with cyclooxygenase inhibitors has been shown to increase pressor responses to infused ANG II in some, but not all, species during pregnancy (29, 59, 151, 152). Similarly, infusion of PGs into intact animals has had variable effects on UBF. For example, systemic infusions of prostacyclin (PGI2) in pregnant ewes
and guinea pigs are associated with a fall in MAP and UBF (26,
111, 151), whereas local intra-arterial infusions have no effect
on MAP but consistently increase UBF (24, 72). In
nonpregnant ewes, PGE2 is a uterine vasodilator, but in
pregnancy it decreases UBF (25, 72). We now appreciate
that many of these contradictory and confusing observations reflect the
mode of administration and the simultaneous effects on MAP or perfusion
pressure and UVR, whereas in other instances this has been due to
increases in myometrial contractility occurring simultaneously with the vascular responses, which obscures vasodilation by increasing intramural pressure (24, 25, 98, 112).
To address these confounding variables, Magness et al.
(79) studied in vitro PGI2 synthesis in intact
uterine and systemic arteries, the latter represented by omental
arteries, at different times in reproduction. Basal PGI2
synthesis by uterine and systemic arteries from near-term pregnant ewes
was 10- and 3-fold greater, respectively, than synthesis by vessels
from nonpregnant animals, and values fell in the postpartum period,
returning to nonpregnant rates by 2 wk postpartum (Fig.
10). Moreover, in pregnancy and the
early postpartum period (~1 wk), basal PGI2 synthesis in
uterine arteries exceeded that by systemic arteries. This difference
was not evident in systemic and uterine arteries collected from
nonpregnant and late postpartum ewes, demonstrating a reversible
pregnancy-induced rise in basal vascular PGI2 synthesis,
which could be involved in the vasodilation and attenuated
vasoconstrictor responses seen in both vascular beds. These
investigators also observed for the first time that incubation of
uterine arteries from pregnant and early postpartum ewes with ANG II
resulted in a further dose-dependent increase in PGI2
synthesis, as reported in the renal and splenic vascular beds
(41, 88). However, the maximum values achieved with
10
5 M ANG II were greatest in arteries from near-term
pregnant ewes, 762 ± 145 vs. 229 ± 47 pg · mg
1 · h
1
(P < 0.001), respectively. This was not seen in
systemic vessels from any group of animals and was inhibited by the
nonspecific ATR antagonist saralasin. Thus a receptor-mediated
mechanism exists in pregnant ovine uterine arteries that has the
potential to further attenuate the vasoconstricting effects of infused
and endogenous ANG II (or other vasoconstrictors) and account for the
differential uterine and systemic responses described
(95). This ANG II-induced rise in uterine artery
PGI2 is derived solely from the endothelium (83,
85), which accounts for ~60% of basal PGI2
synthesis in pregnant uterine and systemic arteries, is mediated by
activating type 1 ATR (AT1R; 32, 86), which are upregulated
in pregnancy (10), and is calcium dependent
(83). Recently, Janowiak et al. (63) reported
that uterine artery endothelium cyclooxygenase-1 is also upregulated in
ovine pregnancy, whereas changes in uterine artery smooth muscle
expression are less clear. Yoshimura et al. (156) confirmed the
stimulatory effects of ANG II on in vitro PGI2 synthesis by
uterine arteries from pregnant ewes, but reported that this effect was
not evident in the maternal placental vasculature, which had
PGI2 and PGE2 synthesis rates that were only
25-30% of that seen in the uterine artery. Glance et al.
(50) observed a similar lack of effect of ANG II on PG
synthesis in the human placenta. The markedly attenuated placental
responses to ANG II, therefore, may not be due to local basal or
stimulated PG synthesis. In preliminary studies, ANG II also increased
human uterine artery PGI2 synthesis (unpublished
observations), but this requires additional study.

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Fig. 10.
Comparison of the in vitro basal production of
6-keto-PGF1 by nonpregnant (NP), pregnant (P), early
postpartum (E), and late postpartum (L) uterine and omental (systemic)
arteries. The prostacyclin metabolite 6-keto-PGF1 was
measured in vitro by radioimmunoassay. SE are presented.
*P < 0.05, values different from NP and L. [Reprinted
from Magness et al. (79) with permission.]
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If basal uterine artery PGI2 synthesis increases in normal
pregnancy as well as uterine synthesis of other PGs, it is logical to
assume that PGs may modulate basal UBF and uterine vascular sensitivity
to vasoconstrictors. However, indomethacin, a nonspecific cyclooxygenase inhibitor, only transiently alters basal UBF, UVR, and
SVR, with values returning to baseline within 15-20 min despite falling PG levels, whereas meclofenamate has no effect (89, 94,
151). Therefore, several investigators have concluded that the
rise in basal uterine PG synthesis in pregnancy does not regulate basal
UVR and UBF. Alternatively, ANG II-induced increases in uterine artery
PGI2 synthesis plus the increase in basal synthesis associated with pregnancy (79) may protect the
uteroplacental vascular bed from the effects of ANG II or other
vasoconstrictors. This was addressed in intact pregnant ewes by
infusing systemic ANG II in the absence or presence of "local"
intra-arterial infusions of indomethacin, a paradigm that would remove
any confounding systemic effects (84). In the absence of
indomethacin, systemic ANG II infusions increased uterine venous
PGI2 from 192 to 1,044 pg/ml dose dependently
(P < 0.05) with a modest effect on arterial levels due
to the large increase in uterine synthesis (84). Thus in
vitro observations (79) were replicated in intact
conscious animals. Although local indomethacin did not alter basal UBF, UVR, or MAP, uterine venous and venoarterial concentration differences of PGI2 fell ~75%, the latter decreasing from 123 ± 29 to 28 ± 14 pg/ml. Furthermore, ANG II no longer affected
uterine venous PGI2, with values remaining ~50 pg/ml.
However, the ANG II-mediated UVR dose-response curve in the treated
uterine horn was shifted upward and to the left, and UBF now fell at
all systemic doses of ANG II (Fig.
11A). The contralateral
uterine horn was unaffected (Fig. 11B) as was the response
in MAP. Thus uterine vascular responses to systemic ANG II infusions
after local cyclooxygenase inhibition resembled those seen in
nontreated nonpregnant ewes (34). Although this is
consistent with observations by McLaughlin et al. (89), it
differs from that observed by Woods (152) in pregnant dogs. In
those studies, uteroplacental sensitivity to ANG II was unaffected by
PG inhibition with meclofenamate. This might be due to differences in
species or the cyclooxygenase inhibitor used.

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Fig. 11.
Effects of continuous systemic ANG II infusions on the
simultaneous relative changes in uterine perfusion pressure (MAP), UVR,
and UBF before and after local intra-arterial indomethacin (INDO)
infusion ipsilateral (A) and contralateral (B) to
INDO. [Reprinted with permission (84).]
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From these observations it is possible to conclude that total PG
synthesis increases during pregnancy in several species, and uterine
synthesis increases dramatically. However, existing data do not
consistently support the hypothesis that increases in basal PG
synthesis account for the systemic or uterine vasodilation characteristic of pregnancy. There is evidence that PGs may modulate uterine and systemic responses to vasoconstrictors, in particular, responses to systemic infusions of ANG II and
-agonists (8, 27). Importantly, these studies and those noted earlier again demonstrate the importance of examining responses to local and systemic
infusions of inhibitors, PGs, and maybe ANG II, which will be addressed later.
More recently, there has been accumulating evidence that vascular NO
synthase (NOS) activity increases in pregnancy and may play a pivotal
role in the vasodilation and attenuated vascular reactivity associated
with pregnancy (see review, Ref. 133). For example,
systemic and peripheral inhibition of NOS increases responsiveness to
infused ANG II in pregnant but not nonpregnant rats (2, 74,
97), suggesting NOS is upregulated in the peripheral vasculature
in pregnancy and serves in part to attenuate responses to ANG II and
other vasoconstrictors. Uterine artery endothelial NOS also is
elevated in pregnancy and is associated with substantial increases in
uterine cGMP production (86, 119, 133, 153). However, its
role in modulating the >30-fold rise in UBF at term pregnancy is
unclear, because short-term intra-arterial infusions of
N
-nitro-L-arginine methyl
ester, a nonspecific NOS inhibitor, decreased uterine cGMP
synthesis without altering basal UBF (133).
Several investigators recently reported that ANG II stimulates local NO
synthesis, and pretreatment with an NOS antagonist enhances constrictor
responses to infused ANG II (39, 139). Furthermore, this
may be mediated through activation of vascular smooth muscle
AT2R (130, 144). As discussed later, this may be important in the uterine circulation. In ovine pregnancy, ~80% of
uterine artery NOS activity is located in the endothelium (85, 86); in contrast to prostacyclin, this does not differ from that
observed in the omental artery. As an indirect measure of NOS activity,
uterine artery cGMP synthesis increases approximately twofold in the
presence of 50 nM ANG II. Unlike PGI2, this is not unique
to the uterine artery of pregnant animals and is observed in systemic
arteries from both groups. Nonetheless, total cGMP production by
uterine arteries, i.e., basal plus ANG II-stimulated, is 2.5-fold
greater than that by omental arteries, ~1,000 vs. 400 fmol/mg of
tissue weight, respectively, which could contribute to the attenuated
uterine responses to infused ANG II in pregnancy and the differences
between the uterine and systemic responses. In fact, the additive
effects of enhanced PGI2 and NO synthesis in the presence
and absence of ANG II may be important. This, however, has not been
well studied to date and requires verification and testing. It also is
unclear if ANG II directly or indirectly increases uterine artery
endothelial NOS activity (130). It is intriguing, however,
that estrogen upregulates both endothelial NOS in endothelium and
neuronal NOS in smooth muscle of uterine arteries from
nonpregnant ewes within 90 min and after daily exposure (119,
127, 146, 147) and is associated with attenuated systemic pressor responses to infused ANG II (80, 104, 122).
Furthermore, uterine responses to ANG II after estrogen treatment are
strikingly similar to that observed in pregnant animals, i.e., the rise
in UVR is less than the rise in MAP (96), which is
opposite that seen in untreated nonpregnant animals (34).
Thus placental estrogens may be involved in modifying vascular
reactivity in pregnancy.
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ANG II AND RECEPTOR EXPRESSION |
It is now clear that at least two subtypes of the ATR are
expressed in large mammals, AT1R and AT2R,
whereas there are three in the rodent, AT1AR,
AT1BR, and AT2R (7, 13, 60, 61). They are considered members of the seven transmembrane-spanning receptor superfamily, are derived from separate gene products, and have
only 40% homology. Of particular note, the AT2R is located on the X chromosome, whereas the AT1R is on chromosome 3 (60). The AT1R is the predominant subtype in
the adult, is inhibited by the specific antagonist losartan, is G
protein coupled, activates phosphoinositide metabolism and
phospholipase C, mobilizes intracellular calcium, and mediates vascular
smooth muscle contraction as well as most other biologic actions of ANG
II (7, 13, 60). In contrast, the AT2R
predominates in the developing fetus (30, 54, 150), is the
major subtype in fetal and early neonatal vascular smooth muscle
(31), does not appear to interact with G proteins
(12, 31), and, although its function is relatively unclear, it appears to modify AT1R effects and participate
in vascular remodeling (13, 30, 43, 60, 130).
Inasmuch as plasma ANG II is elevated in pregnant women and sheep
(76, 77, 93), it stands to reason that the vascular ATR
would be downregulated in normotensive pregnancy, which would explain
the attenuated pressor responses to infused ANG II. However, in
contrast to the myometrium (30, 131), we (30, 76,
118) and others (15, 105) observed that total
vascular smooth muscle ATR binding density (Bmax) and
affinity were similar in pregnant and nonpregnant animals, and this was
true in both the systemic and uterine vasculature. Although Burrell and
Lumbers (17) also observed no change in ovine aortic ATR
Bmax in pregnancy, uterine artery ATR Bmax rose
from ~20 to ~40 fmol/mg protein, the opposite of that anticipated.
Nonetheless, the absence of ATR downregulation in pregnancy raises
questions regarding the mechanisms regulating ATR expression and
turnover in pregnancy, which remain unanswered. While this is not the
subject of this review, placental steroids may be involved (68,
128). When Cox et al. (32) determined ATR subtype
expression in vascular smooth muscle throughout ovine reproduction, the
AT1R was the predominate receptor in all vascular beds
examined except the uterus, where the AT2R accounted for 75-90% of total binding in uterine artery smooth muscle from
nulliparous, pregnant, postpartum, and nonpregnant ewes. Similar
observations were made in uterine arteries from nonpregnant and
pregnant women (35), again demonstrating the striking
similarity between the two species. The only other adult vascular bed
with AT2R predominance is the rat cerebral vasculature
(145). This lack of change in vascular ATR subtype
expression in pregnancy differs from that in the myometrium of women
and sheep, where total Bmax not only falls but is
associated with AT2R downregulation, resulting in AT1R predominance (30, 35, 131). Burrell and
Lumbers (17), however, reported that AT2R
Bmax rose from <5 fmol/mg protein to ~38 fmol/mg protein
in ovine uterine arteries by term gestation, and although
AT2R accounted for ~60% of total binding at term, only
5% was seen in nonpregnant arteries. AT1R binding density was unaltered. Their study differed in that they used uterine arteries
that were frozen with intact endothelium and analyzed in the absence of
protease inhibitors. It is unclear, however, what accounts for the
discrepancy in the two studies. Furthermore, it is unclear why they did
not see the rise in AT1R expression reported in uterine
artery endothelium in ovine pregnancy (10).
Since AT2R do not mediate smooth muscle contraction
responses (30, 32, 35), yet they account for nearly 85%
of ATR binding in uterine artery smooth muscle, we sought to determine
if this might explain the uterine vascular refractoriness to ANG II in pregnant women and sheep. If so, it would raise important questions regarding the mechanism(s) whereby systemic ANG II infusions increase UVR. Although the majority of studies examining UBF responses to ANG II
used systemic infusions, Clark et al. (26) studied the
effects of both systemic and local intra-arterial ANG II infusions on
UBF and UVR. However, only high doses of the peptide were locally infused, and there were no data given regarding the effects on MAP. We
(34), therefore, performed studies in nonpregnant and pregnant ewes comparing uterine and systemic responses to a wide range
of ANG II doses infused either systemically or locally into the uterine
artery to exclude systemic effects. These doses resulted in arterial
plasma concentrations ranging from physiological, i.e., ~400 pg/ml,
to pharmacologic values, ~2,000 pg/ml. To compare responses,
intra-arterial doses were calculated to attain arterial plasma
concentrations achieved during systemic infusions (93). As
anticipated, systemic ANG II infusions recapitulated previous observations; i.e., uterine and systemic responses were greater in
nonpregnant vs. pregnant ewes, and in pregnant ewes, uterine responses
were less than systemic. In contrast, local intra-arterial ANG II
infusions in nonpregnant and pregnant ewes did not elicit a significant
rise in UVR (Fig. 8) or a fall in UBF (Fig. 9) in the absence of a
systemic pressor response. However, whenever UVR rose and UBF fell
during local ANG II infusions, responses were always delayed and always
followed a rise in MAP (Fig. 12), suggesting that ANG II had to reach the systemic circulation before eliciting a uterine response. Furthermore, the rise in MAP was consistently delayed compared with that seen during systemic ANG II
infusions. This pattern of response resembles that observed in the
initial studies of ANG II, i.e., the fall in UVR always followed the
rise in SVR and MAP (Figs. 2-5). More recently, Lambers et al.
(71) reported vasoconstrictive responses to intra-arterial ANG II infusions in estrogenized nonpregnant ewes. However, the bolus
dose used to show specificity of the response was pharmacologic and is
estimated to result in arterial concentrations >5,000 pg/ml. Furthermore, rises in MAP occurred with all other local doses of ANG II
studied, which are estimated to range from >600 to >6,000 pg/ml.

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Fig. 12.
Representative recordings of simultaneous measurements
of mean arterial pressure, heart rate, and UBF in the right and left
uterine arteries of a pregnant ewe before, during, and after the
continuous intra-arterial infusion of a pharmacologic dose of ANG II,
8.0 ng/ml, via the right uterine artery catheter in a near-term
pregnant ewe. The dose was used to characterize delays in pressor
responses and subsequent falls in blood flow associated with local ANG
II infusions. [Reprinted with permission (34).]
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We interpreted our results to mean that the effects of ANG II on the
uterine vascular bed may be mediated by the systemic release of another
more potent vasoconstrictor. This is supported by the predominance of
AT2R binding in uterine vascular smooth muscle and studies
demonstrating that ANG II may enhance catecholamine release (16,
106), delay catecholamine reuptake at the neuromuscular junction
(18), and/or stimulate synthesis and release of smooth muscle endothelin (19). While studies are underway to
examine this, preliminary evidence supports involvement of another
agent (33). Alternatively, simultaneous AT2R
activation may attenuate or inhibit AT1R-mediated increases
in UVR. This is supported by recent studies in nonpregnant estrogenized
ewes and in vitro studies with uterine arteries from pregnant sheep and
rats (71, 90, 157). We also observed ANG II-mediated
constriction of uterine artery rings, which is inhibited by the
AT1R antagonist losartan; however, the responses were quite
small compared with KCl and
-stimulation. We did not see
potentiation by AT2R inhibition or stimulation. Additional
studies are warranted to address this aspect of ANG II-mediated effects.
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ANG II AND VASCULAR REACTIVITY: SMOOTH MUSCLE GROWTH |
Although space does not permit a detailed review of this aspect of
the effects of ANG II on the uteroplacental circulation, ANG II is
known to mediate vascular smooth muscle hypertrophy via the
AT1R (21, 102). In in vitro studies of denuded
uterine artery strips from nonpregnant, pregnant, and postpartum sheep, Annibale et al. (3) were unable to elicit reproducible
responses to ANG II, which is consistent with the observation of
AT2R predominance in these vessels (32).
However, responses to KCl and
-agonist stimulation were enhanced in
uterine arteries from pregnant vs. nonpregnant sheep, which was no
longer evident in the postpartum period. In contrast, renal and carotid
artery responses were unaffected by pregnancy. This is consistent with
other studies examining the difference between uterine artery responses
to ANG II and
-agonists (53, 81, 99, 117, 126).
Annibale et al. (4) subsequently reported that the uterine
artery was hypertrophied in pregnancy and contained increased myosin
and actin contents, consistent with observations by Griendling et al.
(57). St. Louis et al. (136) reported similar
increases in responsiveness to agonists by arcuate arteries from
pregnant rats. They concluded that the mechanical properties of these
arteries had changed. Growth and hypertrophy also occur in more distal
uterine arteries from pregnant rabbits and guinea pigs (23, 66,
67, 101). Thus the uterine vascular bed undergoes substantial
growth and remodeling, resulting in uterine artery hypertrophy, but the
mechanism for this is unclear. It could be due to the rise in UBF and
increase in shear stress, increases in circulating ANG II via
AT1R activation, or the increase in local synthesis of
placental estrogens. Obviously, this is an area that deserves further attention.
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SUMMARY |
Maintenance and growth of the uteroplacental circulation is
essential for the normal growth and well-being of the developing fetus,
and prolonged decreases in UBF result in fetal growth restriction, which may also impair fetal tolerance of labor. The RAS is believed to
play an important role in modulating cardiovascular adaptation during
pregnancy. The development of refractoriness to the vasoconstrictor effects of ANG II is considered an important aspect of this adaptation, because its absence is associated with maternal cardiovascular disease
and increases in fetal growth restriction and fetal and neonatal
morbidity. In this review I have examined the mechanisms whereby the
effects of ANG II on the uterine circulation are normally modulated.
Existing data suggest that the predominance of AT2R binding
in uterine vascular smooth muscle may be the predominant mechanism
responsible for the attenuated uterine responses to infused ANG II in
women and sheep. They also suggest that systemic ANG II infusions may
mediate their effects on the uterine circulation through the release of
other vasoconstricting agents, such as catecholamines. Furthermore, the
combined effects of increases in uterine artery basal and stimulated
PGI2 and NO synthesis in pregnancy may serve to modify
responses to ANG II and these secondary vasoconstrictors. Thus women
with pregnancy-induced hypertension and increased uterine sensitivity
to infused ANG II may have abnormalities in vascular synthesis of PGs
and/or NO, alterations in AT2R function or expression, or
marked increases in these secondary vasoconstrictors, such as
catecholamines. Evidence for the latter is obtained from studies in
women with pregnancy-induced hypertension who appear to have increases
in sympathetic outflow (129, 155). Studies are now
underway to examine this hypothesis. With the recent advent of
genetically engineered models that delete or overexpress various components of the RAS, e.g., angiotensinogen and ATR subtypes, it may
be possible to further delineate the importance of each component in
normal and abnormal pregnancy adaptation.
My thanks to M. Nero who helped in the preparation of this
manuscript, Dr. B. Cox, a valuable collaborator, who provided critical comments on the content and presentation, and to all the people who
have contributed to the ongoing studies in my laboratories.
This work was supported by National Institutes of Health Grant
HD-08783-26 and the George L. MacGregor Professorship in Pediatrics.
Address for reprint requests and other correspondence: C. R. Rosenfeld, Dept. of Pediatrics, UT Southwestern Medical Center at
Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390 (E-mail:
charles.rosenfeld{at}utsouthwestern.edu).