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-induced hypertension in pregnant rats
Department of Physiology and Biophysics and Center for Excellence in Cardiovascular-Renal Research, University of Mississippi Medical Center, Jackson, Mississippi 39216-4505
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Placental ischemia during
pregnancy is thought to release cytokines such as tumor necrosis
factor-
(TNF-), which may contribute to the increased vascular
resistance associated with pregnancy-induced hypertension. We have
reported that a chronic twofold elevation in plasma TNF- increases
blood pressure in pregnant but not in virgin rats; however, the
vascular mechanisms are unclear. We tested the hypothesis that
increasing plasma TNF- during pregnancy impairs
endothelium-dependent vascular relaxation and enhances vascular
reactivity. Active stress was measured in aortic strips of virgin and
late-pregnant Sprague-Dawley rats untreated or infused with TNF-
(200 ng · kg
1 · day1 for 5 days) to increase plasma level twofold. Phenylephrine (Phe) increased
active stress to a maximum of 4.2 ± 0.4 × 103
and 9.9 ± 0.7 × 103 N/m2 in control
pregnant and TNF--infused pregnant rats, respectively. Removal of
the endothelium enhanced Phe-induced stress in control but not in
TNF--infused pregnant rats. In endothelium-intact strips, ACh caused
greater relaxation of Phe contraction in control than in
TNF--infused pregnant rats. Basal and ACh-induced nitrite/nitrate production was less in TNF--infused than in control pregnant rats.
Pretreatment of vascular strips with 100 µM
NG-nitro-L-arginine methyl ester, to
inhibit nitric oxide (NO) synthase, or 1 µM
1H-[1,2,4]oxadiazolo[4,3-]quinoxalin-1-one, to
inhibit cGMP production in smooth muscle, inhibited ACh-induced
relaxation and enhanced Phe-induced stress in control but not in
TNF--infused pregnant rats. Phe contraction and ACh relaxation were
not significantly different between control and TNF--infused virgin
rats. Thus an endothelium-dependent NO-cGMP-mediated vascular
relaxation pathway is inhibited in late-pregnant rats infused with
TNF-. The results support a role for TNF- as one possible
mediator of the increased vascular resistance associated with
pregnancy-induced hypertension.
nitric oxide; cytokines; pregnancy
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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NORMAL PREGNANCY is often associated with a reduction in systemic vascular resistance and arterial pressure and decreased vascular reactivity to circulating vasoconstrictor substances (15, 28, 32, 36). The hemodynamic and vascular changes observed during normal pregnancy have been explained, in part, by increased nitric oxide (NO) synthesis by various cells, including vascular endothelial cells (1, 14, 38, 41, 46). This is supported by reports that the tissue expression and specific actions of NO synthase are elevated during late gestation (3, 9, 40, 45) and that the metabolic production and plasma level of cGMP, a second messenger of NO and a cellular mediator of vascular smooth muscle relaxation (24, 27), are increased during pregnancy (11).
In 5-7% of pregnancies, women develop a condition called
preeclampsia, characterized by increased intravascular coagulation, proteinuria, increased systemic vascular resistance, and
pregnancy-induced hypertension (PIH) (19, 35). Although
PIH is a major cause of maternal and fetal mortality, the mechanisms of
this disorder have not been clearly identified. Because of the
difficulty of performing mechanistic studies in pregnant women, several
animal models of PIH have been developed (2, 4, 7, 12, 13, 16,
28, 30, 33). Studies in these animal models have proposed that a
reduction in the uteroplacental blood flow and the ensuing placental
ischemia during late pregnancy initiate a cascade of hemodynamic and vascular changes that lead to increased systemic vascular resistance and PIH (2, 12, 16, 30). In support of
this hypothesis, we previously found that reduction in uteroplacental perfusion in pregnant rats results in significant hemodynamic changes
and a hypertensive state that closely resembles PIH in women
(2). We also found that the reduction in uteroplacental perfusion pressure in pregnant rats is associated with decreased vascular relaxation and enhanced vascular reactivity of the systemic vessels and suggested that these vascular changes could be the cause of
the increased vascular resistance and hypertension (12). However, it is not clear how a localized reduction in uteroplacental perfusion pressure could lead to generalized vascular changes in the
maternal circulation. For a localized reduction in uterine perfusion pressure to cause generalized vascular changes, one would
predict possible release of vasoactive factor(s) from the ischemic placenta into the systemic circulation. According to the "cytokine" hypothesis of PIH, the reduction in uteroplacental perfusion pressure and the ensuing placental ischemia are
thought to increase the release of cytokines from the placenta into the maternal circulation; the increased plasma cytokines would then lead to
the generalized vascular changes and hypertension (8, 10, 29,
43). In support of the cytokine hypothesis, it has been shown
that the plasma levels of cytokines such as tumor necrosis factor-
(TNF-) and interleukin (IL)-6, which is activated by TNF-, are
elevated nearly twofold in women with preeclampsia (10, 23, 29,
43). Also, we recently found that a two- to threefold elevation
in plasma TNF- in late-pregnant rats results in significant
elevation in vascular resistance and arterial pressure, while elevation
of plasma TNF- to the same level in virgin rats does not cause any
significant hemodynamic changes (22). However, the
vascular mechanisms underlying the TNF--induced increases in
vascular resistance and arterial pressure in pregnant rats are still unclear.
The present study was designed to test the hypothesis that a two- to
threefold elevation in plasma level of TNF-, produced by infusing
the cytokine into chronically instrumented late-pregnant rats at a rate
to mimic the plasma levels observed during PIH in preeclamptic women
(29, 43), is associated with decreased endothelium-dependent vascular relaxation and increased vascular reactivity. We used virgin and late-pregnant rats to investigate 1) whether the vascular reactivity is enhanced in
TNF--infused pregnant rats compared with control pregnant rats,
2) whether endothelium-dependent vascular relaxation is
inhibited in TNF--infused pregnant rats compared with control
pregnant rats, and 3) whether the reduction in vascular
relaxation and enhancement of vascular reactivity in TNF--infused
pregnant rats involve alterations in the endothelium-dependent NO-cGMP pathway.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals.
Female virgin (nonpregnant; 12 wk, ~200-250 g) and time-pregnant
(day 12 of gestation, ~350 g) Sprague-Dawley rats were
purchased from Harlan Sprague Dawley (Indianapolis, IN). The rats were
housed individually in the animal facility and maintained on ad libitum standard rat chow and tap water in a 12:12-h light-dark cycle. The rats
were divided into 4 groups of 12 rats each: virgin control, pregnant
control, virgin TNF--infused, and pregnant TNF--infused. On
day 14 of gestation or the equivalent in virgin rats, all
rats were anesthetized with isoflurane and underwent a surgical
procedure for catheter implantation. A section of PE-50 tubing was
placed in the carotid artery for measurement of arterial pressure and blood sampling. The catheter was filled with heparin and exteriorized at the back of the neck. The rats were also instrumented with a venous
catheter and a miniosmotic pump. TNF--treated rats were infused
intravenously with TNF- (Biosource, Camarillo, CA) at a rate of 200 ng · kg1 · day1 for 5 days
to increase plasma levels approximately twofold. Control rats were
infused with normal saline. Rats were then housed individually, allowed
to recover, and studied 5 days later (day 19-20 of
pregnancy or the equivalent in virgin rats). All procedures were
performed in accordance with the guidelines of the Animal Care and Use
Committee at the University of Mississippi Medical Center and the
American Physiological Society.
Measurement of mean arterial pressure in conscious rats. On the day of the experiment, each rat was placed in a Plexiglas restrainer. The carotid arterial catheter was connected to a Statham pressure transducer, and the mean arterial pressure was continuously recorded on a Grass polygraph (model 7D, Astro-Med, West Warwick, RI).
Measurement of plasma TNF-.
On the day of the experiment, blood samples (0.5 ml) were collected for
measurement of plasma TNF- in control and TNF--infused virgin and
pregnant rats using a rat TNF- ELISA system (Cytoscreen, Biosource).
This assay is a solid-phase sandwich-type system that utilizes a
specific anti-rat TNF- antibody coated onto the wells of microtiter
plates. Serum samples (50 µl) and standards were pipetted in
triplicate into appropriate microtiter wells, and the assay was
performed according to the manufacturer's instructions. The
sensitivity of this TNF- ELISA system is 0.7 pg/ml, and the upper
limit of detection is 150 pg/ml. The average recovery of TNF- in
serum pools from normal rats is 97%. No significant cross-reactivity was noted with a battery of other human and murine cytokines. With the
use of this protocol, the plasma levels of TNF- were found to be
6.7 ± 2.2 and 13.5 ± 1.8 pg/ml in control and
TNF--infused rats, respectively.
Tissue preparation. On the day of the experiment (day 19-20 of pregnancy), the rats were anesthetized by inhalation of isoflurane. The thoracic aorta was rapidly excised, placed in oxygenated Krebs solution, and cleaned of connective tissue. The aorta was cut transversely into 3-mm-wide rings. Aortic rings were cut open into strips. For endothelium-intact vascular strips, extreme care was taken throughout the procedure to avoid injury of the endothelium. For endothelium-denuded vascular strips, the endothelium was removed by gentle rubbing of the vessel interior with wet filter paper.
Isometric tension. A thread loop was used to attach one end of the vascular strip to a glass hook, and the other end was connected to a Grass force transducer (model FT03, Astro-Med). Vascular strips were stretched to maximum length (Lmax, i.e., 1.5 times the unloaded initial length). Lmax was measured separately in vascular strips of virgin and pregnant rats. Lmax in virgin rats was not significantly different from that in pregnant rats. Vascular strips were allowed to equilibrate for 1 h in a water-jacketed, temperature-controlled tissue bath filled with 50 ml of Krebs solution continuously bubbled with 95% O2-5% CO2 at 37°C. The changes in isometric tension were recorded on a Grass polygraph (model 7D, Astro-Med).
A control contraction was elicited by applying 105 M
phenylephrine (Phe) to the tissue bath solution. Once the Phe
contraction reached a plateau, the tissue was rinsed three times for 10 min each with Krebs solution. The whole procedure of contraction and washing was repeated twice. Increasing concentrations of Phe were applied, the contractile responses were recorded, and
concentration-response curves were constructed.
In other tissues, a contraction to submaximal concentration of Phe was
elicited. Increasing concentrations of ACh or sodium nitroprusside were
added, and the extent of vascular relaxation was measured. In other
experiments, the tissues were pretreated for 30 min with 100 µM
NG-nitro-L-arginine methyl ester
(L-NAME) to inhibit NO synthase or with 1 µM
1H-[1,2,4]oxadiazolo[4,3]quinoxalin-1-one (ODQ) to inhibit cGMP production in smooth muscle (25), and the
effects on the Phe-induced contraction and on ACh-induced relaxation of the Phe contraction were observed.
Nitrite/nitrate production.
Endothelium-intact vascular strips were placed in test tubes containing
1.5 ml of Krebs solution aerated with 95% O2-5%
CO2 at 37°C, and the solution was changed every 10 min
for 1 h. Samples for basal accumulation of nitrite formed from
released NO were first taken. The Krebs solution was replaced, and the
strips were stimulated with ACh for 10 min. The vascular strips were
rapidly removed, dabbed dry with tissue paper, and weighed. The
incubation solutions were assayed for the stable end product of NO,
NO
Solutions, drugs, and chemicals. Normal Krebs solution contained (in mM) 120 NaCl, 5.9 KCl, 25 NaHCO3, 1.2 NaH2PO4, 11.5 dextrose, 1.2 MgCl2, and 2.5 CaCl2 at pH 7.4. Stock solutions of L-phenylephrine HCl, ACh, bradykinin, sodium nitroprusside, and L-NAME (Sigma) were prepared in distilled water. ODQ (Calbiochem, La Jolla, CA) was dissolved in dimethyl sulfoxide (final concentration <0.1). All other chemicals were of reagent grade or better.
Statistical analysis.
The developed force was corrected for the cross-sectional area of each
individual strip and expressed as active stress (N/m2)
using the following equation: stress = force/cross-sectional area,
where cross-sectional area = wet weight/(tissue density × length of the strip) and tissue density = 1.055 g/cm3.
Data were analyzed and expressed as means ± SE, with n
representing the total number of experiments (12-16)
performed on individual vascular strips isolated from six to eight
different rats of each group. Data were compared using ANOVA
with multiple classification criteria [rat type (pregnant vs. virgin),
condition of endothelium (intact vs. denuded), and treatment (control
vs. chronically infused with TNF- or untreated vs. acutely treated
with L-NAME or ODQ)] followed by Bonferroni's post test
to compare selected groups or Dunnett's post test to compare all
groups with the control group. Differences were considered
statistically significant if P < 0.05.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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On the day of the experiment (day 19-20 of
gestation or the equivalent in virgin rats), the mean arterial pressure
was 96 ± 3 mmHg in control pregnant rats and was significantly
elevated in TNF--infused pregnant rats (123 ± 3 mmHg). In
contrast, the mean arterial pressure was not significantly different
between control virgin rats and TNF--infused virgin rats: 107 ± 4 and 109 ± 3 mmHg, respectively.
In endothelium-intact vascular strips of control pregnant rats, Phe
caused concentration-dependent increases in contraction (Fig.
1A). The Phe-induced
contraction appeared to be greater in TNF--infused pregnant rats
(Fig. 1B) than in control pregnant rats (Fig. 1A)
but did not appear to be different between control virgin rats (Fig.
1C) and TNF--infused virgin rats (Fig. 1D). To
correct for the difference in the size of the vascular strips, the Phe
contraction was normalized for the cross-sectional area of the vascular
strip and presented as active stress (see METHODS). The Phe
concentration-active stress curve in TNF--infused pregnant rats was
enhanced compared with that in control pregnant rats (Fig.
2A). The maximal Phe-induced
active stress was significantly greater in TNF--infused pregnant
rats than in control pregnant rats (Table
1). Removal of the endothelium
significantly enhanced the Phe-induced stress in control pregnant rats
but caused slight and insignificant increase in Phe-induced stress in
TNF--infused pregnant rats (Fig. 2A, Table 1). In
contrast, the Phe-induced active stress was not significantly different
between control virgin rats and TNF--infused virgin rats (Fig.
2B). When the Phe response was presented as a percentage of
the maximum Phe contraction and the Phe ED50 was
calculated, Phe was more potent in causing contraction in
endothelium-denuded than in endothelium-intact vascular strips of
control pregnant rats (Fig. 2C, Table 1). In contrast, Phe
was only slightly more potent in causing contraction in
endothelium-denuded than in endothelium-intact strips of
TNF--infused pregnant rats (Fig. 2C, Table 1). The Phe
response as a percentage of maximum was not significantly different
between control virgin rats and TNF--infused virgin rats (Fig.
2D).
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In endothelium-intact vascular strips, pretreatment with 100 µM
L-NAME for 30 min, to inhibit NO synthase, significantly enhanced the Phe-induced stress in control pregnant rats (Fig.
3A, Table 1). Also, plotting
of the Phe response as a percentage of maximum and calculation of the
Phe ED50 showed that Phe was more potent in causing
contraction in L-NAME-pretreated than in untreated vascular
strips of control pregnant rats (Fig. 3C, Table 1). In
contrast, the maximal Phe-induced stress and the Phe ED50
were not significantly different between L-NAME-pretreated
and untreated vascular strips of TNF--infused pregnant rats (Fig. 3,
B and D, Table 1).
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Similarly, in endothelium-intact strips, pretreatment with 1 µM ODQ
for 30 min, to inhibit cGMP production in smooth muscle (21,
25), enhanced Phe-induced stress in control pregnant rats (Fig.
3A, Table 1). Also, Phe was more potent in causing contraction in ODQ-pretreated than in untreated vascular strips of
control pregnant rats (Fig. 3C, Table 1). In contrast, the maximal Phe-induced stress and the Phe ED50 were not
significantly different between ODQ-pretreated and untreated vascular
strips of TNF--infused pregnant rats (Fig. 3, B and
D, Table 1).
In endothelium-intact vascular strips of control pregnant rats, ACh
caused concentration-dependent relaxation of submaximal Phe (6 × 107 M)-induced contraction (Figs.
4A and
5A). Because the Phe
contraction in other groups of rats was greater than that in control
pregnant rats, the Phe concentration was adjusted in the
TNF--infused pregnant rats (3 × 108 M), control
virgin rats (3 × 107 M), and TNF--infused virgin
rats (3 × 107 M) to produce a submaximal
contraction that is roughly equal in magnitude to that in control
pregnant rats. The ACh-induced relaxation of Phe contraction was
reduced in TNF--infused pregnant rats compared with control pregnant
rats (Figs. 4B and 5A). Also, when the
ACh-induced response was presented as a percentage of the maximal
ACh-induced relaxation, ACh was less potent in inducing relaxation in
TNF--infused pregnant rats than in control pregnant rats:
ED50 = 1.2 ± 0.06 × 106 and
0.6 ± 0.04 × 107 M, respectively. ACh-induced
relaxation was not significantly different between control virgin rats
and TNF--infused virgin rats (Figs. 4, C and
D, and 5B). Pretreatment of endothelium-intact strips with 100 µM L-NAME or 1 µM ODQ significantly
inhibited the ACh-induced relaxation of Phe contraction in control
pregnant rats (Fig. 6A) but
not TNF--infused pregnant rats (Fig. 6B). Removal of the
endothelium completely inhibited the ACh-induced relaxation of Phe
contraction in all groups of rats.
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In endothelium-intact vascular strips of control pregnant rats, the
basal nitrite/nitrate production was 42.1 ± 7.5 pmol/mg tissue
weight, and ACh caused concentration-dependent increases in
nitrite/nitrate production (Fig. 7). The
basal and ACh-induced nitrite/nitrate production showed significant
reduction in TNF--infused pregnant rats compared with control
pregnant rats (Fig. 7). The basal and ACh-induced nitrite/nitrate
production were not significantly different between control virgin rats
and TNF--infused virgin rats (Fig. 7).
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In endothelium-denuded vascular strips of all groups of rats, sodium
nitroprusside, an exogenous NO donor and a standard guanylate cyclase
activator (24), caused concentration-dependent relaxation of Phe contraction. However, no significant differences in the magnitude of sodium nitroprusside-induced relaxation of Phe contraction were observed between control and TNF--infused pregnant (Fig. 8A) or virgin rats (Fig.
8B).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The main findings of the present study are as follows:
1) the mean arterial pressure in late-pregnant rats with
twofold elevation of plasma level of TNF- is significantly greater
than that in control pregnant rats, 2) vascular reactivity
is greater in TNF--infused pregnant rats than in control pregnant
rats, 3) endothelium-dependent vascular relaxation is
less in TNF--infused pregnant rats than in control pregnant rats,
4) the activity of the endothelium-dependent NO-cGMP
pathway is reduced in TNF--infused pregnant rats compared with
control pregnant rats, and 5) the arterial pressure,
vascular reactivity, and vascular relaxation are not significantly
different between control virgin rats and TNF--infused virgin rats.
Consistent with previous studies from our laboratory and others, we
have found that the mean arterial pressure and the vascular reactivity
to various vasoconstrictors are reduced in pregnant rats compared with
virgin rats (13, 15, 28). The present study has also shown
that the mean arterial pressure and the vascular reactivity to the
-adrenergic agonist Phe are enhanced in TNF--infused pregnant
rats compared with control pregnant rats. The findings in the
TNF--infused pregnant rats are consistent with previous studies,
which have shown that the arterial pressure and the vascular reactivity
to vasoconstrictors are enhanced in other animal models of hypertension
during late pregnancy (7, 12, 13, 28). In search of the
possible mechanisms involved in the observed enhanced vascular
reactivity in TNF--infused pregnant rats, we found that removal of
the endothelium significantly enhanced the Phe contraction in normal
pregnant rats but had minimal effects in TNF--infused pregnant rats.
Also, the ACh-induced relaxation was less in TNF--infused pregnant
rats than in normal pregnant rats. These results suggest that an
endothelium-dependent relaxation pathway is intact in control pregnant
rats but is possibly impaired during elevation of plasma TNF- in
late-pregnant rats.
One important vasodilator released from endothelial cells is NO
(20, 26, 34, 37). The reduced ACh-induced relaxation in
TNF--infused pregnant rats could be due to a decrease in the synthesis and release of NO from endothelial cells or a change in the
sensitivity of vascular smooth muscle to relaxation by NO. The
sensitivity of vascular smooth muscle to relaxation by NO could be
evaluated by its sensitivity to relaxation by exogenous NO donors such
as sodium nitroprusside. The observation that relaxation of
endothelium-denuded vascular strips by sodium nitroprusside was not
significantly different between control and TNF--infused pregnant
rats provided evidence that the endothelium-independent mechanisms of
vascular relaxation and the sensitivity of vascular smooth muscle to
relaxation by NO are not impaired in TNF--infused pregnant rats and,
thereby, suggests that the impaired ACh-induced relaxation in
TNF--infused pregnant rats is most likely due to a decrease in the
synthesis and/or release of NO from endothelial cells.
To further investigate the possible role of NO synthesis and release in
the proposed impaired endothelium-dependent relaxation pathway in
the TNF--infused pregnant rats, we found that pretreatment of the
vascular strips with L-NAME, which is known to block NO synthesis, significantly inhibited vascular relaxation by ACh and
enhanced the vascular reactivity to Phe in control pregnant rats but
had minimal effects in TNF--infused pregnant rats. These results
suggest that NO synthesis by endothelial cells is intact in normal
pregnant rats but is impaired during elevation of plasma TNF- in
late-pregnant rats. This is further supported by the observation that
the basal and the ACh-induced nitrite/nitrate production were
significantly reduced in vascular strips of TNF--infused pregnant
rats compared with control pregnant rats.
The NO produced by endothelial cells is known to promote vascular
relaxation by activating guanylate cyclase and increasing cGMP
production in vascular smooth muscle (24, 26). We found that ODQ, which is known to inhibit guanylate cyclase and to decrease cGMP production in smooth muscle (21, 25), significantly
inhibited the endothelium-dependent vascular relaxation by ACh and
enhanced the vascular reactivity to Phe in endothelium-intact strips of control pregnant rats but not TNF--infused pregnant rats. These results further support the contention that NO production or release by
endothelial cells and, thereby, the activity of the NO-cGMP pathway in
vascular smooth muscle are reduced in TNF--infused pregnant rats
compared with control pregnant rats.
The present results support the hypothesis that placental
ischemia contributes to maternal endothelial cell dysfunction
by enhancing the synthesis of cytokines such as TNF- and IL-1
(8, 10, 23). The data are also consistent with the reports
that the plasma levels of TNF- and IL-6, which is activated by
TNF-, are elevated nearly twofold in women with preeclampsia
(10, 23, 29, 43). The effects of TNF- appear to be
dependent on the plasma levels of the cytokine. Although high levels of TNF-, as observed during septic shock or after administration of a
high dose of lipopolysaccharide (LPS), activate gene expression of
inducible NO synthase, modest levels of TNF- have been shown to
downregulate the mRNA of endothelial NO synthase (47).
This is consistent with a recent study by Faas and co-workers
(18) that showed that intravenous infusion of a high dose
of the endotoxin LPS, which is known to activate TNF-, decreases
blood pressure in conscious pregnant rats, while a very low-dose
infusion of LPS results in significant and long-term increase in blood
pressure and urinary albumin excretion and significant platelet
aggregation (18).
It is important to emphasize the following cautionary remarks regarding
the above interpretations. First, although the present results suggest
that the decrease in endothelial cell function and the increase in
vascular reactivity observed in the TNF--infused pregnant rats could
contribute to the observed increase in blood pressure, these results
should be interpreted with caution, since the changes in endothelial
cell function and vascular reactivity may also be secondary to blood
pressure elevation. Analysis of the time course of the changes in
vascular reactivity and the increase in blood pressure should help
determine whether the relationship between these two parameters is
causal or associative in nature. Second, the chronic effects of TNF-
in vivo could be due to a direct effect on the vascular endothelium or
perhaps indirect effects through the release of other factor(s).
Although a recent report suggests that direct exposure to TNF- and
IL-1 causes functional alterations in endothelial cells
(39) and reduction in ACh-induced vasodilation and
relaxation of vascular strips of normal male rats (44),
whether these direct effects of TNF- on endothelium-dependent
vascular relaxation are altered in females, particularly during
pregnancy, remains to be investigated. Third, the vascular endothelium
has been shown to release other vasodilator substances, in addition to
NO, such as endothelium-derived hyperpolarizing factor and prostacyclin
(5, 42). This may explain why, in the vascular strips of
TNF--infused pregnant rats, some relaxation to ACh was still
observed and was not completely inhibited by L-NAME or ODQ.
On the other hand, the complete absence of ACh-induced relaxation in
endothelium-denuded strips of TNF--infused pregnant rats still
supports the contention that the ACh-induced relaxation is endothelium
dependent. Fourth, although the present results provided evidence that
the enhanced vascular reactivity in the TNF--infused pregnant rats
may involve inhibition of an endothelium-dependent NO-cGMP pathway, we
cannot rule out the possibility that an increase in the release of
contracting factors from the endothelium or an increase in the
sensitivity of vascular smooth muscle to endothelium-derived contracting factors also occurs. This is supported by reports that
long-term inhibition of NO synthesis during mid- to late gestation in
rats is associated with elevated plasma levels of endothelin-1
(17) and that TNF- and IL-1 stimulate the production of
endothelium-derived contracting factors including endothelin-1 (31). This is also supported by the present observation
that removal of the endothelium or pretreatment of vascular strips of
control pregnant rats with L-NAME or ODQ caused an
enhancement of Phe-induced vascular reactivity to levels that were
still less than that observed in the TNF--infused rats. These
observations suggest that the elevation of plasma TNF- during late
pregnancy in rats may be associated with additional alterations in the
cellular mechanisms of vascular smooth muscle contraction and should
represent interesting areas for future experiments. We should note that the causes of the lack of effects of TNF- in virgin rats and its
dramatic vascular effects in pregnant rats are unclear but could be
related, in part, to the plasma levels of sex hormones such as estrogen
and progesterone and possible synergistic actions of the sex hormones
on the vascular effects of TNF-. This is supported by reports that
the plasma estrogen and progesterone levels are elevated during
pregnancy and are higher in pregnant than in virgin rats
(2). This is also supported by in vitro studies that have
shown that estradiol enhances leukocyte binding to TNF--stimulated
endothelial cells via an increase in TNF--induced adhesion molecules
(6). Studying the effects of acute and long-term exposure
to estrogen and progesterone on the vascular effects of TNF- should
help further identify the mechanisms underlying the possible
synergistic interactions between sex hormones and the cytokine and
should represent important areas for future investigations.
In conclusion, the present results suggest that an
endothelium-dependent relaxation pathway involving the production and
release of NO from endothelial cells and increased cGMP production in smooth muscle is inhibited in systemic vessels of pregnant rats chronically treated with TNF-. The results suggest a role for TNF- as one possible mediator of the increased vascular resistance associated with pregnancy-induced hypertension.
Perspectives
The search for the cellular and vascular mechanisms underlying the hypertension induced in pregnant animal models should help us understand better the pathophysiological basis of preeclampsia in pregnant women. Abnormal reduction in uteroplacental blood flow during late pregnancy has been suggested as an initiating event that triggers a cascade of events leading to increased vascular resistance and hypertension. The present results suggest that the release of cytokines such as TNF- from the
ischemic placenta is one factor that may lead to endothelial
cell dysfunction, decreased vascular relaxation, and thereby increased
vascular resistance and pregnancy-induced hypertension. However, the
present data represent the observed changes in endothelium-dependent
vascular relaxation at one specific point in time, namely, day
19 of pregnancy in rats and after 5 days of TNF- infusion.
Future time course studies should be useful to identify the time of
onset and the time to peak changes in endothelium-dependent vascular
relaxation in TNF--infused pregnant rats and to determine whether
these vascular changes precede or coincide with the changes in the
arterial pressure. We should also note that the observed vascular
effects of TNF- in pregnant rats may not be limited to only this
cytokine. Placental ischemia contributes to maternal
endothelial cell dysfunction by enhancing the synthesis of not only
TNF- but also IL-1. Also, the plasma levels of the cytokine IL-6 are
elevated almost twofold in women with preeclampsia. Whether chronic
infusion of other cytokines such as IL-1 or IL-6 during late pregnancy
would produce vascular effects similar to those of TNF- remains to
be investigated. In relation to this question, it is not clear whether
the observed chronic effects of TNF- represent direct vascular
effects of the cytokine or may be mediated by other factors.
Interestingly, TNF- has been shown to activate IL-6. Therefore,
studying the acute vascular effects of not only TNF- but also other
cytokines such as IL-6 should help further delineate the role of
cytokines as possible mediators of pregnancy-induced hypertension.
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ACKNOWLEDGEMENTS |
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This work was supported by a grant-in-aid from the American Heart Association, Mississippi Affiliate, and National Heart, Lung, and Blood Institute Grants HL-33849, HL-51971, and HL-52696.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. A. Khalil, Dept. of Physiology and Biophysics, University of Mississippi Medical Center, 2500 North State St., Jackson, MS 39216-4505 (E-mail: rkhalil{at}physiology.umsmed.edu).
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.
10.1152/ajpregu.00270.2001
Received 8 February 2001; accepted in final form 18 September 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Endo) strips were incubated in normal Krebs
solution and then stimulated with increasing concentrations of Phe. Phe
contraction was measured and presented as active stress
(A and B) or as percentage of maximum Phe
contraction (C and D). Data points represent
means ± SE of measurements in 12-16 vascular strips from
6-8 rats of each group.
