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Introduction
Cardiovascular disease ranks among the leading causes of morbidity and mortality in adult populations in most countries
in the world. Significant progress in understanding the etiology of cardiovascular disease has come from recent recognition
that inflammation plays a key role in its
development[1]. Most of the studies now focus on the role of inflammation in some
cardiovascular diseases, such as
atherosclerosis[2], and ischemic heart
disease[3] or heart
failure[4,5]. Among these diseases, hypertension is a major risk factor and its underlying pathogenetic mechanisms are still not well elucidated. What we know
about hypertension is only that its pathogenesis is a complex and multifactorial phenomenon affected by genetic predisposition,
metabolism, and environment. Now there is accumulating clinical evidence showing that inflammation is also related to
hypertension to some extent, which was also discovered in some cardiovascular diseases. Sesso and
colleagues[6] reported a positive relationship between increased serum levels of the C-reactive protein and the risk of development of incident
hypertension in participants of the Women's Health Study. A total of 20525 women were followed up prospectively for a
median of 7.8 years, during which time approximately one fourth of the women acquired elevated blood pressure. Those with
higher levels of the C-reactive protein were more likely to develop hypertension. As we know, C-reactive protein levels in the
upper ranges of the normal distribution are widely believed
to reflect a state of low-grade chronic inflammation. Therefore,
the association between higher C-reactive protein levels and
new-onset hypertension led us to focus our attention on the
relationship between inflammation and hypertension. We
wondered whether inflammation participated in the
pathogenesis of hypertension.
Bautista et al[7] discovered an elevated level of
inter-leukin-6 (IL-6) in a study of hypertensive patients.
Mean-while, Chae et al[8] found the relationship between this
cytokine level and the values of blood pressure. More
interestingly, Samuelsson[9] showed that prenatal exposure
to IL-6 resulted in hypertension and increased
hypothalamic-pituitary-adrenal (HPA) axis activity in adult rats. It has been
commonly accepted that IL-6 is a pro-inflammatory factor
which is produced by various cells of the organism,
including monocytes, macrophages, fibroblasts, and endothelial
cells. Thus, prenatal exposure to IL-6 might induce maternal
systemic inflammation[10]. Moreover, systemic inflammatory
response during pregnancy represents a form of stressful
event for the fetus. Nevertheless, there are some data
suggesting that IL-6 possesses anti-inflammatory
properties[11]. Is hypertension in offspring induced single-handedly by IL-6
or by maternal systemic inflammation? This hypothesis has
not been tested. Therefore, we chose lipopolysaccharide
(LPS) from Gram-negative bacteria to act as a non-specific
immunostimulant to induce maternal systemic inflammation.
LPS has been shown to stimulate the HPA axis via the release
of cytokines including IL-6[12,13]. Prenatal exposure to LPS
results in increased basal plasma corticosterone levels and a
reduction in the number of central glucocorticoid receptors,
which are important factors contributing to the regulation of
blood pressure. Therefore, we speculated that prenatal
exposure to LPS would probably cause hypertension in
offspring. One probable way through which inflammation
caused hypertension might be via maternal inflammation in
utero, which can affect the development of the offspring
organs.
Materials and methods
Animals Nulliparous, time-mated Sprague-Dawley (SD)
rats were purchased from the Animal Center of the Third
Military Medical University (Chongqing, China). All the rats
had ad libitum access to both standard laboratory rat chow
and tap water and were caged individually in a room under
constant temperature (24 °C) and a 12 h/12 h light/dark cycle
until parturition. Pups were raised with a lactating mother
until 4 weeks of age, and thereafter they lived in cages with
3 rats per cage. All surgical and experimental procedures
were carried out in accordance with institutional animal care
guidelines.
Prenatal LPS exposure After 1 week of acclimation, the
dams were randomly divided into 2 groups. Each group
contained 8 pregnant rats. On their 8th, 10th, and 12th
gestational days, the dams (n=8) received ip injections of 0.79
mg/kg LPS (Escherichia coli 026:B6, Sigma, St Louis, MO,
USA) dissolved in 1 mL sterile saline. The control dams
(n=8) received sterile saline only. Gestation lasted for
20_22 d. Two treated dams and 1 control dam did not deliver
any pups. After birth, the litters were counted and weighed.
The pups were, after recording the birth weight, randomly
chosen to be included in this study (males: controls,
n=12; LPS-treated, n=12; females: controls,
n=12; LPS-treated, n=12) within 1 week. All of the chosen pups were redistributed
within the same treatment group of dams so that each group
had 3 males and 3 females per lactating mother. They were
left undisturbed until 4 weeks of age when they were weaned.
Blood pressure measurement Arterial blood pressure
was measured in conscious rats using the tail-cuff method
(ML125, PowerLab, ADInstruments, Castle Hill,
Australia)[14]. Before the measurement, the animals were placed inside a
warming chamber (about 34 °C) for 15 min. The aim of
the procedure was to calm the animals and dilate the tail
blood vessels. Then the rats were placed in plastic restrainers.
It was ensured that Perspex restraint cages were selected to
fit the animal comfortably. The rats were placed in the Perspex
cylinder restraint cage and the depth was adjusted forwards
and backwards within the tube to restrict movement. The
tube was kept in a proper position to prevent the animal from
turning around. A cuff with a pneumatic pulse sensor was
attached to the tail and was positioned at the proximal end of
the tail. The rats were allowed to habituate to this procedure
for 7 d before the experiments. The active site of the pulse
transducer was located on the ventral surface of the tail,
directly below the caudal artery. The transducer was
positioned directly following the tail cuff. Maximum sensitivity
was achieved when the artery was positioned above the most
sensitive position on the transducer. Systolic blood
pressure (SBP) appeared when the cuff pressure corresponded
to the restoration of the first caudal artery pulse. Arterial
blood pressure was measured at least 3 times for each animal.
Body weight The offsprings' body weights were
regularly monitored once every 2 weeks during the experiments
from the age of 4 to 24 weeks.
Food intake When the rats reached 15 weeks of age,
food consumption for each cage was recorded once a day.
Each cage contained 3 rats which were presented with the
same amount of food, and their intake was measured the
following day by subtracting the uneaten food. This was
done during 1 week and was calculated as food intake in
grams per rat per day as well as in grams per body weight per
day.
Adipose tissue weight At the end of the 24th week, the
rats were killed by decapitation. The epididymal, parametrial,
mesenteric, retroperitoneal, inguinal, and around the kidney
adipose tissues were rapidly excised and weighed.
Serum level of leptin The blood samples were obtained
by decapitation. Then they were allowed to clot for half an
hour at room temperature before centrifuging at
3000×g for 20 min. The serum was removed and stored at -20 °C until
radioimmunoassay (Linco Research Company, St Charles,
Missouri, USA).
Statistical analysis All data were expressed as mean±
SD. The means were evaluated by Student's unpaired
t-test. P<0.05 was considered to be statistically significant.
Results
Dams and litters There were no significant differences
in the number of progeny per dam [9.7±3.6 (5_15) and
9.0±2.9 (5_13) pups/dam for treated (n=6) and control
(n=7) dams, respectively; P=0.72]. Meanwhile, no
significant differences were discovered for the ratio of male births
to total births in each litter [LPS-treated, 0.51±0.08 (0.40_
0.60); controls, 0.49±0.11 (0.40_0.67);
P=0.61]. The body weights of the newborn pups did not differ much between
the LPS and control groups [male pups: 6.1±0.9 (4.8_7.7) and
6.3±0.8 (5.1_7.7) g; P=0.67; female pups: 6.0±0.8 (4.7_7.4) and
6.2±0.7 (5.1_7.6) g; P=0.26].
Blood pressure measurement It was found that all
offspring with prenatal exposure to LPS had elevated SBP from
the age of 6 to 24 weeks. As the offspring rats grew older,
the blood pressure of those with prenatal exposure to LPS
began to rise and was higher than that of the controls. The
difference between these 2 groups become more significant
(123.40±9.07 vs 109.35±5.89 mmHg, 120.66±5.52
vs 108.98±
4.21 mmHg, male and female rats, respectively, compared
with the same gender controls at the end of 20th week;
P<
0.01), although the SBP in all offspring had not reached the
standard level of hypertension in SD rats (Figure 1).
Body weight In the male offspring, the total body weights
had statistical significance since the age of 6 weeks (114.95±
4.92 g vs 107.95±7.19 g; P<0.05). However, there was a
statistically significant difference since the age of 14 weeks in the
female offspring (230.47±7.94 g vs 212.86±7.66 g;
P<0.01) (Figure 2).
Food intake The food intake of each rat increased in
15-week-old male LPS offspring compared with the control
offspring [23.4±1.6 (21.6_25.9) and 19.6±1.0 (18.2_21.0)
g/day, respectively; P<0.01]. The food intake of each female
offspring was [19.0±1.3 (17.6_20.9) and 14.7±1.1 (13.2_16.0)
g/day, respectively; P<0.01]. The food intake, measured by
per body weight per day, in the male offspring was [0.075±
0.005 (0.067_0.085) and 0.066±0.002 (0.063_0.073)
g·d-1·g-1 bodyweight, respectively;
P<0.01] and that of the female offspring was [0.072±0.009 (0.061_0.087) and 0.064±0.004
(0.057_0.070)
g·d-1·g-1 bodyweight, respectively;
P<0.05](Figure 3).
Adipose tissue weight The total weight of various tissues,
including epididymal, retroperitoneal, mesenteric, inguinal,
and around the kidney fat depots in male offspring and
parametrial instead of epididymal fat depots in female
offspring 24 weeks of age are shown in Table 1. All the fat
depots weight in the abdomen were significantly heavier in
LPS offspring, regardless of sex, than that of the controls
(P<0.01).
Serum level of leptin The serum level of leptin was
significantly higher in offspring with prenatal exposure to
LPS than that of the controls (P<0.01) both in males (2.30±
0.19 ng/mL vs 1.34±0.16 ng/mL, respectively) and females
(1.91±0.15 ng/mL vs 1.32±0.16 ng/mL, respectively; Figure
4).
Discussion
Hypertension is a major risk factor for cardiovascular
disease, so effective control of hypertension is an important
goal of cardiovascular therapies. Current evidence supports
a central role for inflammation in all phases of
atherosclerosis[15]. The recognition that atherosclerosis is a special case
in the general category of inflammation provides a tool to
unify and simplify the understanding of complex processes.
The possibility that hypertension, at least in part, is a
product of arterial pathology similar to that of atherosclerosis is
intriguing. It was thus suggested that there might be a
correlation between inflammation and hypertension. Some
researchers reported that prenatal exposure to LPS could
result in obesity and insulin resistance in adult male rat
offspring[16]. As we know, obesity and insulin resistance are
risk factors for hypertension. We noticed that LPS could
alter maternal immune systems and induce maternal
inflammation[17]. Maternal LPS exposure can also result in the
existence of cytokines in the amniotic fluid and
corticotrophin-releasing hormone in the fetal rat
brain[18]. Thus, maternal inflammation may participate in the pathogenesis of
hypertension in adult rat offspring. In order to test such a
hypothesis, we chose LPS, which acted as a non-specific
immunostimulant and intraperitoneally injected it to
pregnant rats. The dosage of LPS we chose could induce
systemic inflammation, resulting in a low percentage of fetal
anomalies, but not abortion[19]. In addition, the rationale for
choosing time phages on gestational d 8, 10, and 12 was that
this period was in the second trimester, a period of early fetal
brain development[20].
The present study found that prenatal exposure to LPS
led to increases in blood pressure that might potentially
develop into hypertension in male and female offspring. This
phenomenon has not been reported elsewhere until now.
The mechanisms of increases in blood pressure should be
related to maternal inflammation induced by LPS. Maternal
systemic inflammation may serve as a stressful event for
fetus development and can induce high blood pressure in
adult offspring.
We also found that the offspring (regardless of sex) with
prenatal exposure to LPS showed hyperphagia and increases
in body weights and abdominal fats weight when they grew
up. All of the chosen pups were redistributed within the
same treatment group of dams when they were born. Each
group consisted of 3 males and 3 females per lactating
mother. Therefore, the offspring in either the LPS group
or the control group had the same pattern of lactation.
However, the offspring in the LPS group still showed
heavier body weight since the age of 6 weeks in males and
at the age of 14 weeks in females, and hyperphagia in the
15th week compared with those in the control group. This
hyperphagia and increases in body weight in the offspring
might have been induced by maternal inflammation as well
as increases in blood pressure. The male offspring became
fat earlier than the female ones, which might be due to the
difference in sex. Meanwhile, the basic blood pressure in
the LPS offspring was higher than that of the control
offspring since 6 weeks of age. However, body weight showed
no change in LPS female offspring as compared with the
control offspring. In the male offspring with prenatal
exposure to LPS, the blood pressure had little change
compared with that of the control group. However, the body
weight in the LPS offspring increased more than that of the
control group. Thus, increases in blood pressure were body
weight-independent in offspring with prenatal exposure to
LPS. Increases in body weight induced by maternal
inflammation was perhaps only a risk factor in the development of
hypertension[21].
Meanwhile, the serum level of leptin in both males and
females was elevated in the LPS group than in the control
group. Leptin was produced by adipose tissue and its
receptors were localized in the hypothalamic area in the
brain[22]. The increased food intake, despite higher levels of
leptin in serum, showed that the regulation of food intake by
leptin was inefficient after maternal immune challenge. Leptin,
also considered a pro-inflammatory cytokine that belongs to
a family of long-chain helical cytokines and has structural
similarity with IL-6, prolactin (a growth hormone), plays an
important role in inflammatory processes and immune
responses. The increase in leptin production that occurred
during infection and inflammation strongly suggests that
leptin is a part of the cytokine network that governs the
inflammatory-immune response[23]. Thus, in the LPS
offspring, the higher levels of leptin implied that the
offspring might have been in an inflammatory and
immune-stimulated state.
In conclusion, our results have demonstrated that
prenatal exposure to LPS results in increases in blood pressure
that might develop into hypertension in adult rats. This
discovery may serve to modify our current strategy and
medical interventions for the prevention or treatment of
cardiovascular disease. The exact mechanisms underlying the
development of increases in blood pressure and body weight
in the LPS offspring remain unclear, so further studies should
be conducted to determine the mechanisms of how maternal
systemic inflammation results in increases in blood pressure
and body weight in offspring.
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