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Introduction
Pulmonary arterial hypertension (PAH) is a common
disease induced by many etiological factors. Pulmonary
vascular remodeling by monocrotaline (MCT) affects both
vascular smooth muscle and vascular wall connective tissue at
the molecular level[1]. Endothelium plays an important role
in controlling vascular tone as well as vascular remodeling
by releasing endothelium-derived relaxing factors [EDRF,
nitric oxide (NO)] and contracting factors mainly
endothelin-1 (ET-1)[2]. ET-1 is a potent endothelium-derived
vasoconstrictor peptide, which possesses mitogenic effects to proliferate
the vascular smooth muscle cells (VSMC) in the
development of PAH[3]. NO, which is released from the endothelium,
attenuates proliferation of VSMC[4] and is opposed
functionally to ET-1[5].
Monocrotaline (MCT), a pyrrole alkaloid, causes
substantial and sustained inflammatory damage to the
pulmonary arterial system and, therefore, causes PAH in
rats[5]. CPU 86017, p-chlorobenzyltetrahydroberberine (Figure 1), a
new compound derived from berberine, possesses a calcium
channel blocking effect[6], combined with an antioxidative
effect[7]. CPU 86017 ameliorates the progression of chronic
congestive heart failure in relation to suppression of the
ET-1 system[8]. Therefore, it is hypothesized that CPU 86017
might relieve the pulmonary hypertension, with an indirect
effect on the overactive ET pathway underlying the
pulmonary hypertension induced by MCT. We intend to explore
the pathogenesis of PAH induced by MCT in relation to an
activated ET pathway, and its downstream events (iNOS and
ROS activity) in response to the medication of CPU 86017.
Materials and methods
Experimental protocol Sixty-five male Sprague-Dawley
rats (weighing 250 g-300 g, from the Experimental Animal
Center of Nanjing Medical University) were randomized into
5 groups (n =13). Animals (apart from the normal group)
were injected with a single dose 60 mg/kg, sc)
of MCT (Promega). p-Chloro-benzyl-tetrahydro-berberine chloride
(CPU 86017) was first synthesized by the New Drug Center
of China Pharmaceutical University and provided by
Shan-dong Xinhua Pharmaceutical Industry Co. The 5 groups
were the normal group (Ctl), PAH untreated group (PAH), and
the 3 treatment groups (CPU 86017 20, 40, and
80 mg·kg-1·d-1
,po, daily for 28 d from the day of MCT, sc). CPU 86017 was
suspended freshly in 0.5% CMC-Na of 0.4%, 0.8%, and 1.6%
(20, 40, and 80
mg·kg-1·d-1, sc). The normal and PAH groups
were given an equal volume of CMC-Na.
The investigation was performed in accordance with the
Guide for the Care and Use of Laboratory Animals in Jiangsu
Province.
Hemodynamic analysis and heart weight index
After 4 weeks of MCT medication and interventions with CPU 86017,
rats were killed with urethane (1.5 mg/kg, ip). RVSP (right
ventricular systolic pressure), CVP (central venous pressure),
and blood pressure were measured using 2 catheters
separately, as described previously[9]. Animals were then
exsanguinated, and the hearts and lungs were dissected and
weighed. The right ventricular remodeling was assessed by
measuring the RVWI (right ventricle weight index): the RV
weight to body weight ratio (RVW/BW, mg/g) and the
RV-to-left ventricular (LV) plus septal (S) weight ratio
[RVW/(LVW+SW), mg/g]. The improvement after drug
intervention was calculated as follows: improvement
% = (PAH-Treated)/(PAH-Normal)×100%.
ET-1, NO, NOS, superoxide dismutase (SOD), and
malondialdehyde (MDA) assays Blood samples were
collected in chilled vials containing 30 mL EDTA2Na and
20 mL Aprotinin. ET-1, NOS activity, SOD, and MDA in serum and
lung tissue were measured using radioimmunoassay or the
kits supplied by Nanjing Jiancheng
Biotechnological Co, following the practice described
previously[9].
Assessment of pulmonary artery remodeling
Transverse sections of lungs, 4 µm in thickness, were cut at the right
middle zones and stained with hematoxylin-eosin, then the
wall thickness of small pulmonary arteries (diameter<150 µm)
was analyzed using a colorful picture analysis system
(Quantment 500), as described in a previous
report[9]. The thickness (µm) and WT (wall thickness ratio) percentage
value were determined as the average data of 10 fields per
slice and the WT was calculated as below:
WT%=(diameterext-diameterint)/diameter
ext×100.
mRNA expressions of preproET-1, iNOS, and eNOS in
lung tissue Lung tissue was homogenized in TRIZOL, and
then extracted with hydroxybenzene-chloroform. Reverse
transcription-polymerase chain reaction (RT-PCR) was
performed in 25 µL final volume under the following conditions:
initial denaturalization at 94 oC for 5 min, denaturalization at
94 oC for 40 s, annealing for 40 s at 64
oC for preproET-1, at 58 oC for eNOS, at 58
oC for iNOS, and at 60 oC for
b-actin, all extension at 72 oC for 1 min. The circular
numbers of b-actin, preproET-1, eNOS, and iNOS were 25, 28, 26 and 28,
respectively. The sequences of primers are shown in Table 1
and the procedures were the same as described
previously[9]. The PCR products were electrophoresed and determined
semi-quantitatively.
Functional assessment of isolated pulmonary arterial
rings The pulmonary arteries were carefully isolated and
cleaned of outer connective tissue in cold K-H solution satu
rated with 100% oxygen. The rings from each pulmonary
artery (approximately 3 mm in length) were mounted
vertically between 2 hooks in organ chamber myographs, which
were filled with K-H solution and kept at 37
oC. Isometric tension was measured with force transducers (MPA-V).
Constrictions of the isolated pulmonary arterial rings by KCl
100 mmol/L or phenylnephrine 1 µmol/L were performed in
calcium free KH solution and after adding up to 2.5 mmol/L
Ca2+ in the presence of either KCl or phenylephrine,
separately[10-12].
Statistical analysis Data are presented as mean±SEM.
For statistical evaluation one-way analysis of variance was
used, following DennettĄ¯s test. The Student Newman Keuls
test was performed when the variance was equal, and the
Games-Howell test was performed when variance was not
equal. Pearson correlation analysis was also carried out on
some indices. A value of P<0.05 was considered statistically
significant.
Results
Hemodynamic changes The RVSP and CVP in MCT PAH
group were elevated markedly by 79% (P<0.01) and 2400%
(P<0.01) compared with normal, respectively.
CPU 86017 20, 40, and
80 mg·kg-1·d-1
po decreased the RVSP by -18.5%, -37.2%, and -15.1%, compared to the PAH
(P<0.01), respectively, and the reduction in CVP was by -66.7%, -73.3%, and -80.0%,
respectively (P<0.01, Table 2).
Regression in RVWI The RVWI in the control group
was increased by 50.0 % in RVW/BW and 53.3 % in
RVW/(RVW+SW) (P<0.01), respectively. Reduction in
RVW/BW by CPU 86017 20, 40, and
80 mg·kg-1·d-1
, po was -30.5%, -33.3%, and -25.9%,
respectively, compared to the untreated group and in RVW/(RVW+SW) was -28.2%, -30.4%, and
-28.2% (P<0.01), respectively. The remodeling of the right
ventricle was regressed remarkably in each intervention
group (Table 3).
Regression in the vasculature of the pulmonary arteriole
In PAH group, the thickness (mm) and the relative value of
WT% (wall thickness percentage) of small pulmonary
arteries (<150 µm in diameter) were increased by
106.0% and 80.7% (P<0.01) compared with the control group. In CPU 86017 80,
40, and
20 mg·kg-1·d-1
groups, thickness (mm) and the relative value of WT percentage of small pulmonary arteries
decreased significantly compared with the untreated group
(Table 4, Figure 2).
NO, ET-1, cNOS, SOD, and MDA in serum and lung
tissue In the PAH group, the ET-1 in the serum and lungs
was dramatically increased by 53.9% (P<0.01) and 93.7%
(P<0.01), respectively. CPU 86017 decreased the content of
ET-1 to the normal level in lung tissue, but was less effective
in serum. The NO content was increased by 60.0%
(P<0.01) in serum, but decreased by -52.5%
(P<0.01) in pulmonary tissue, compared to that of the normal group. The level of NO
was significantly increased in CPU 86017 80 and
40 mg·kg-1·d-1
groups in tissue, whereas the difference was not significant
in serum. Activity of cNOS in tissue decreased by -38.5%
(P<0.01) in the PAH group compared to the normal. The
activity of cNOS was increased by 33.0%
(P<0.01) in CPU 86017
80 mg·kg-1·d-1
po group compared to the PAH (Table 5,
6). The MDA was significantly increased in both the serum
and lungs, whereas the activity of SOD decreased markedly.
A significant reduction in MDA production was observed in
all three CPU 86017 groups, which was associated with an
increase in the SOD activity in the serum and lungs.
PreproET-1, iNOS, and eNOS mRNA expression
Twenty-eight days after MCT injection, the mRNA
expression of preproET-1, eNOS, and iNOS in the PAH group was
upregulated by approximately 4, 6.5, and 2.7 times,
respec-tively, against the normal lung tissue. The upregulation of
eNOS and iNOS mRNA levels was significantly reversed in
three CPU 86017 groups, and preproET-1 mRNA abundance
was also reduced notably in CPU 86017
80 mg·kg-1·d-1
group versus the PAH group (Figure 3).
Isolated pulmonary artery activity Two sorts of
pulmonary vascular activities were assessed
using high KCl and phenylephrine in separate experiments. Each was evaluated
in 2 phases of constriction of the isolated pulmonary artery
rings in calcium-free medium, and up to 2.5 mmol/L calcium
was added to the calcium-free medium in the presence of
either high KCl or phenylephrine. The KCl induced
vasoconstrictions in the calcium-free medium decreased
markedly in the PAH group and recovered partially after
CPU 86017 intervention. The constrictions by the calcium
influx on adding calcium into calcium-free medium in the
presence of KCl 100 mmol/L were impaired markedly in the
untreated group, whereas there was no improvement in the CPU
86017-treated groups (Figure 4). The contractions by
phenylephrine in the calcium-free medium were notable in the
untreated group, and no improvement could be found after
CPU 86017 treatments. A markedly impaired vascular tone of
contractions by calcium influx resulting from an addition of
Ca2+ into the medium in the presence of phenylephrine 1
mmol/L was found and vascular tension completely returned
to normal in the CPU 86017 groups of 80 and 40
mg·kg-1·d-1,
po (Figure 5).
Discussion
CPU 86017 is derived from chemical modification of the
moiety of berberine and possesses a blocking effect on the
K+ and Ca2+
channels[13], together with an improvement in
the water solubility and availability in pharmacokinetics. The
chemical reformation leads to a reinforcement of calcium
antagonism, providing a potential beneficial use in the
treatment of cardiovascular
disorders[12].
The chronic inflammation of the lung tissue by MCT
contributes to an insult to vascular endothelium and myocardium,
consequently resulting in a substantial increase in ET-1
release, which promotes the pathological progression of
PAH. The voltage-dependent calcium channels are coupled
to ETA receptors by G proteins; therefore, an enhancement of
the calcium influx is anticipated to follow an excess ET-1
release, as we found in the PAH. The activation of the
L-type Ca2+ channels caused by an excess ET-1 in the
myocardium and vasculature is an important etiological factor
resulting in maladjustment and remodeling changes in the
pulmonary vessels and the right ventricle.
The ET-1 closely linked with the ROS system is an
important pathway involved in cardiovascular
derangement[14]. One of the up-stream events in the pathway to the tissue ET-1 is
the mRNA of preproET-1, which is also elevated significantly.
An excess of ET-1 binding to the ETA receptors stimulates
the protein kinase C (PKC), mitogen-activated protein
kinase (MAPK) pathway, finally leading to an abnormality of
phenotype of genes encoding the preproET-1, iNOS, and
cNOS, matrix formation, as well as to pulmonary VSMC
proliferation[15-17].
A low level of NO and elevated ET-1 in the pulmonary
tissue is a result of endothelial damage and dysfunction by
MCT. MCT selectively damages pulmonary endothelium,
reducing NO production and secretion. The vascular NO is
biosynthesized and released in a constant manner, which is
necessary to maintain the normal vascular vasodilatation
response. The bioavailability of NO (ie basal release) is
compromised significantly, resulting in impaired vascular
activity in the PAH group. An abnormally low NO exerts a
suppressive effect on the proliferation of smooth muscle cells,
because of an excess of ET-1, which mediates an activation
of MAPK pathway. An increased serum NO in the PAH
group is a result of activated activity of the iNOS resulting
from the stimulation of chronic inflammation by MCT, which
accelerates both pulmonary artery hypertension and portal
hypertension[18].
The ROS play an important role in remodeling of the small
pulmonary artery, as they proliferate smooth muscle cells by
activating the phosphorylation of ERK/MAPK. The ET-1
stimulates the downstream events in the pathway; that is,
activation of iNOS and ROS. It is likely that a circulated
activation mechanism takes place between the upstream and
the downstream to exacerbate an activation of the ET
pathway involved in the pathological process. An increment of
ROS causes an increase in the abundance of mRNA of
prepro-ET-1, eNOS, and iNOS, attributable to the enhancement of
the transcription process in the
nucleus[19,20]. This is easily reversed by suppression of the ET-ROS pathway after
treatment with CPU 86017.
An intervention to interrupt the targets located at either
the upstream or the downstream of the ET pathway is
capable of relieving the disturbance in the PAH by MCT. The
insult to the pulmonary endothelium and the later developed
myocardial insufficiency of the right ventricle are the main
factors contributing to excess ET-1 and the over-stimulation
of the ET pathway. The overactivated ET pathway can be
reversed indirectly by the calcium antagonism and
antioxida-tive effects of CPU 86017. Actually an attenuation of the
activated ET pathway by CPU 86017 in the MCT PAH in rats
can not be ascribed to a direct blocking action on ET receptors,
but ascribed to a calcium antagonism relieving an injury of
the pulmonary endothelium and the myocardium and
rebalancing an interruption of the ROS formation. A reduced
formation of the ET-1 and an indirect blockade of endothelin
receptors to alleviate its downstream events are achieved
using CPU 86017 chronic medication.
Plasma ET-1 was less responsive than the pulmonary
origin ET-1to CPU 86017 treatment. The ET can be sourced
from the two: physiological and pathological conditionas,
sush as the ET in the affected lung is totally from the insult
from MCT. In the blood stream the majority of ET is released
from the organ under normal conditions such as the central
nervous system, the kidney, endometrium, and normal
tissue[21], but ET from the affected lung contributes to a small
portion of plasma ET-1. So only a heavy inhibition of the
release from the pathological lung by the high dose of CPU
86017 could provide a reduction in the seral total ET, but not
the middle and small doses. In contrast, paracrine and
autocrine ET-1 is effective in the local region. The
pulmonary origin of ET-1 was the main pathological factor and
responded well to the CPU 86017 treatment.
We found that eNOS mRNA expression was significantly
increased in MCT-induced PAH, and was up to 6.5 times
higher than that in normal group, whereas both NOS activity
and NO content decreased in the same way as reported by
Resta et al[22]. Such a contradiction might be contributed to
damage to endothelium by MCT with which the eNOS can
not be converted into a normally active form. The
maladjustment resulted from phosphorylation of
threonine[23], which
causes defective activity of the eNOS, contributing to PAH
progression[24].
In conclusion, an activated ET-ROS pathway damages
the endothelium system which results in consequent
pulmonary vasculature derangement in PAH by MCT in rats. By
suppressing the ET pathway, CPU 86017 relieves MCT PAH.
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