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Introduction
Smooth muscle cells (SMC) play a very important role in
the development of the cardiovascular system in human
fetuses. The arterial duct, which connects the systemic and
pulmonary circulations, is derived from the aortic arch and
normally extends from the main or left pulmonary artery to
the descending aorta just distal to the origin of the left
subclavian artery. The ductus is usually 5_10 mm long before
birth, exhibiting some length variability, and the diameter
varies from a few millimeters to 1_2 cm. Closure of the
ductus occurs at or after birth during the transition from fetal to
postnatal circulation. Isolated patent ductus arteriosus
(PDA) occurs approximately once in every 2500_5000 live
births. The incidence of PDA is related to several factors,
including decreased smooth muscle content in the ductal
wall, diminished responsiveness of the ductal smooth muscle
to oxygen, and possibly other factors. Postnatal ductus
constriction produces hypoxia of the inner vessel wall, which
might lead to cell death and the release of some
hypoxia-inducible growth factors that stimulate endothelial
proliferation. Permanent closure of the ductus arteriosus
requires loss of cells from the muscle media through the
programmed cell death pathway and the development of
neointimal mounds by proliferation of endothelial
cells[1]. Thus, failure in the constriction of the postnatal arterial
ductus could be the key factor for the event of PDA. Some
pathways are considered to be associated with controlling
cell apoptosis and proliferation. Apoptosis is regulated by
proteins of the Bcl-2 family consisting of both anti-apoptotic
and pro-apoptotic factors. Bax regulates
apoptosis[2] by binding to Bcl-2, and the Bax expression level over Bcl-2 is thus
considered an indicator for cell apoptosis or growth.
Mitogen activated protein kinases (MAPK) regulate cell growth
and apoptosis, and much attention has focused in recent
years on 3 subfamilies of MAPK: the extracellular
signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and
the p38 MAPK[3]. These enzymes are regulated by a
characteristic phosphorylation system in which a series of 3
protein kinases phosphorylate and activate one
another[4]. The ERK function controls cell division, and the inhibition of
these enzymes is generally considered antiproliferative in
certain cells[5,6]. The JNK are critical regulators of
trans-cription, and JNK inhibitors may contribute to apoptosis
resistance.
Retinoic acid (RA), an oxidative metabolite of vitamin A,
regulates many biological processes, including embryonic
development and cell
differentiation[7_9]. Vitamin A is considered an important factor for heart development. There
are suggestions that there is a link between vitamin A and
PDA[10], and RA has been shown to be important in the
development of the fetal ductus
arteriosus[11]. In this study, we investigated whether human fetal SMC from the aorta,
pulmonary artery, and arterial duct tissues possess
different biological behaviors, vulnerabilities to teratogenic
insults, and distinct response to RA. We investigated the
RA receptor antagonist influence on the effect of RA.
Materials and methods
Samples A total of 8 human fetal embryonic samples
were obtained from Wenzhou Medical College (Wenzhou,
China) under Chinese law statutes. The tissue collection
used in the current research was obtained with the approval
of the local Human Ethics Committee (Wenzhou Medical
College, China), and our procedures were in compliance with
the Helsinki Declaration on human experimentation. All
specimens were obtained from voluntary pregnancy terminations
due to personal reasons other than health problems, was
and were induced by mifepristone and prostaglandins;
written informed consent was obtained from all donors. The
gestational periods varied from 18_22 weeks of development
with an average 18±2 weeks. The embryonic age was
determined by measuring the head diameter and crown-rump
length based on the data published by Forutan et
al[12] and calculated according to the time from the last menstrual
period (LMP) under the assumption that fertilization occurs
14 d after the LMP. No abnormal data were found in the
obstetrical examination of the conceived women and the
B-supersonics examination of the fetus before the operation.
Main reagents Dulbecco's modified Eagle's medium
(DMEM) and fetal bovine serum (FBS) were from Gibco
BRL (Carlsbad, CA, USA). The immunohistochemistry SP
kit was from Zymed (South San Francisco, CA , USA).
Proliferating cell nuclear antigen (PCNA), antihuman
α-actin(smooth muscle), and antihuman Bax monoclonal antibody
were from Neomarkers (Fremont, CA, USA). Antihuman
Bcl-2 monoclonal antibodies were from Zymed (USA).
Fast-activated, cell-based extracellular (FACE)
signal-regulated kinases 1/2 and JNK ELISA kits were from
Activemotif (Carlsbad , CA, USA). RA was from
Sigma_Aldrich (St Louis, MO, USA). CD2366 was kindly
provided by R&D (Sophia, Antipolis, France). The enhanced
chemiluminescent (ECL) horseradish peroxidase (HRP)
substrate was from Millipore (Billerica, CA, USA).
Primary tissue culture After the pregnancy terminations,
the fetus samples were immediately kept on ice under
asepsis condition. Within 2 h after the operation, the fetal heart,
aorta, and pulmonary artery were dissected. Primary fetal
SMC (VSMC) from the aorta, pulmonary artery, and arterial
ductus were cultured within 24 h after induction of cervical
dilation by the modified explant-attached
method[13]. Briefly, under a microscope, the aorta, pulmonary artery, and arterial
ductus were isolated. All the vessels were vertically cut and
the intimal epithelium tissue was scrapped by a
scalpel. Then the vessels were washed by cool DMEM culture medium
and cut into 0.5_1 mm3 pieces. The pieces were covered and
pressed with glass slides in 60 mm diameter culture dishes
and maintained in DMEM containing 20% FBS at 37 oC with 5% CO2 so that cells could grow on the surface of both the
dish bottom and cover glass. The double surface enabled
the cells to grow well on both sides within a short period.
Half the medium was replaced with fresh DMEM containing
20% FBS 3 times a week until the cells were confluent around
the tissue pieces. The cells on the glass slides were used for
the staining of SMC, and the cells in the dishes were
continuously cultured.
RA induction Fetal SMC from the aorta, pulmonary artery,
and arterial duct tissues from fewer than 5 passages were
prepared on glass slides. When the cells adhered onto the
slides, all slides were placed in fresh DMEM containing 20%
FBS. 0.5 µmol/L RA was added to the RA group,
0.5 µmol/L CD2366 was added to the CD group, and
0.5 ìmol/L RA and CD2366 was added to the RA+CD group. The medium
containing the same volume of ethanol was set as the control
group. All cells were incubated for 24 h and removed from
the medium for the observation of PCNA, Bax, and Bcl-2
protein expressions. All of the experiments were repeated at
least 5 times.
Immunohistochemistry Immunohistochemistry was
performed according to the manufacturer's protocol of the
immunohistochemistry SP kit. Briefly, PCNA, Bax, and Bcl-2
antibody concentrations were 1:100, 1:100, and 1:100,
respectively. The α-actin antibody specific for SMC was
diluted at 1:200. HRP-conjugated goat antimouse
immunoglobulin was used as the secondary antibody and worked at
1:1000. Mouse immunoglobulins replaced the first
antibodies on the negative control slides. The samples were
visualized by diaminobenzidine tetrahydrochloride, the HRP
substrate, and observed under an Olympus light microscope
(Tokyo, Japan). Positive staining was shown as brown or
yellow. The staining intensity of the color was classified
into 3 grades: dark brown (+++), light brown (++), and yellow
(+). The positive signal of each case was quantified as the
number of cells with +++ ×3, ++ ×2, and + ×1, as determined
by weighing. On each slide, 200 SMC were randomly chosen,
observed, and counted for the positive staining percentage.
Western blotting The cells were washed twice with
phosphate-buffered saline (PBS) and lysed in buffer consisting
of 50 mmol/L Tris-HCl (pH 8.0), 0.5% (v/v) Tween 20, 1
mmol/L EDTA, 2 mmol/L phenylmethylsulfonyl fluoride, and 1
µg/mL aprotinin. The proteins were assayed by the Bradford method
and mixed equally with 2× SDS loading buffer and resolved
on SDS_PAGE gel. The proteins on the gel were then
transferred onto a polyvinylidene difluoride membrane (Millipore,
Bedford, MA, USA). After blocking with 5% milk protein in
PBST (PBS containing 0.05% Tween 20) for 2 h at room
temperature, the membranes were incubated with specific
primary antibodies for 6 h at 4 oC. Then the blots were washed
3 times with PBST and incubated for 2 h with
HRP-conjugated secondary antibodies. Immunolabeling was detected
by ECL after washing with PBST. All experiments were
repeated at least 3 times independently.
Flow cytometric analysis The flow cytometric analysis
method was according to our previous
study[9]. The cells were collected and washed 3 times with 10 mL PBS (pH 7.4)
after culture. Then the cell pellets were added with 1 mL
hypotonic solution (50 µg/mL propidium iodide, 0.1%
sodium citrate, and 0.1% triton X-100) at 4 oC overnight. Then 20 000 cells were acquired and detected in the FACS (Becton
Dickinson Biosciences, Mountein View, CA, USA) machine
by Cell Quest software (Becton Dickinson Biosciences,
Mountein View, CA, USA). The data of the cell cycle were
analyzed by Mod Fit LT version 2.0 software fitting the
apoptosis mode.
FACE ELISA The FACE signal-regulated kinase
measurement was according to the protocol provided by the
manufacturer (Activemotif, USA). Briefly, for the detection
of activated (phospho-) ERK1/2 and JNK in different media, the
cultured primary aorta, pulmonary artery, and arterial duct cells
from fewer than 5 passages were prepared in
5×105cells/mL, respectively, and 100 µL cell suspension was added to each
well of the 96-well plate. After all of the cells had been
incubated for 48 h and adhered to the bottom of the
wells, 100 µL cultured media containing 20% FBS (as the control group),
0.5 µmol/L RA, 0.5 µmol/L CD2366, and 0.5
µmol/L RA+CD2366 were added to the wells as 4 groups,
respectively, replacing the original media. After treatment,
the cells in the 96-well plate were fixed with 100 µL of 4%
formaldehyde in PBS. Relative phospho-ERK1/2 and phospho-JNK levels were detected with a cell-based ELISA
kit. The phospho-ERK1/2 and phospho-JNK antibodies were
applied into the well at concentrations of 1:250 and 1:125,
respectively. PBS replaced the antibodies in the negative
control wells. After incubation, the addition of the
secondary antibody, and washing, chromogen was added. After
development, the absorbance of each well was read on a
spectrophotometer within 5 min at a wavelength of 450 nm
over 655 nm, which indicated the expression level of
phospho-ERK1/2 and phospho-JNK. Meanwhile, the absorbance of each well was also read at 595 nm after crystal
violet staining, which indicated the cell numbers in each well.
The absorbance values at 450 nm were normalized according
to the absorbance value at 595 nm.
Statistics The data in the study were presented as
mean±SD for each group. ANOVA and the
SNK(Student-Newman-Keuls) test were used for the variance analysis
among the test groups. Comparisons between 2 groups were
analyzed by Student's t-test.
Results
Characteristics of the fetal SMC After 3_5 d of primary
culture, the cells were observed growing from the edges
around the tissues and in emitting order. After a 2 week
incubation period, many long, spindle-like cells were
observed. The third passage cells from the tissues were
prepared for staining with the monoclonal antibody against
a-actin, which is specific for SMC. The results found that
97% of the cells were stained positive, and the control
showed negative staining by mouse immunoglobulins (Figure
1A). The cells from the aorta, pulmonary artery, and arterial
ductus tissues were all 95%_97% positive with α-actin. We
also tested this antibody with a 39 week embryonic lung
tissue and did not find it to stain (data not shown). This
suggested the cultured cells were fetal SMC.
Next, we examined the expression of PCNA on fetal SMC
from these 3 tissue sources by immunostaining and Western
blotting. The results shown in Figure 1B suggest that the
cells from the arterial ductus expressed the lowest level of
PCNA among the 3 groups in 6 cases. There was a significant
variance among the 3 groups (P<0.01). Bax/Bcl-2 was
considered as a switch for controlling cell growth or
apoptosis[14,15]. In these 3 kinds of primary cells, the SMC from the arterial
ductus were found to have the highest expressional ratio of
Bax/Bcl-2 (1.04:1). In the aorta and pulmonary artery SMC,
the Bax/Bcl-2 ratio was 0.55:1 and 0.629:1, respectively.
Interestingly, we also observed that the expression levels of
PCNA and Bcl-2 were related to fetal age. As the embryos
grew, the arterial duct SMC expressed less PCNA and Bcl-2
(Figure 1C). The value for ERK was also found to decrease
with age.
Response to RA We further observed the response of
these primary SMC to RA exposure. Interestingly, RA did
not suppress arterial duct SMC growth, but stimulated the
cells to express PCNA. In the control cells, the relative
signal intensity for PCNA was 142.67±12.08. After RA treatment,
the signal intensity was enhanced to 175.33±10.80
(P<0.05, n=6; Figure
2A,2B,2G). Together with CD2366, a RA
receptor-selective antagonist[16], the induction of PCNA by RA
expression was overcome. There were no differences of the
PCNA expression levels among the control, CD and RA+CD
groups. The treatment with only CD2366 failed to enhance
PCNA expression. In the aorta or pulmonary artery SMC,
RA treatment did not induce any significant change of PCNA
expression.
In contrast, RA treatment significantly inhibited Bax
expression (Figure 2C,2D,2G) in the arterial duct SMC, and
the relative signal intensity decreased to 100.00±12.31
from 196.71±25.79 in the control
(P<0.01, n=6). Such inhibition of
Bax expression could not be overcome by adding CD2366
because CD2366 itself reduced Bax expression. However,
Bcl-2 expression after RA treatment as reduced slightly from
the control 189.33±21.13 to 162.83±20.2. Therefore, the ratio
of Bax/Bcl-2 increased to 1.21:1 from 1.04:1, which suggested
that RA stimulated proliferation in the primary fetal arterial
duct SMC. In the aorta and pulmonary artery SMC, RA
treatment did not significantly increase the ratio of Bax/Bcl-2
(Figure 2E,2F,2G). In this study, treatment with 0.5 µmol/L
RA for 24 h did not cause any significant apoptosis peak in
the flow cytometric analysis.
Activation of ERK1/2 and JNK ERK can be activated by
phosphorylation of the molecule. The phospho-ERK was
thus considered the activated form. Therefore, we further
investigated the expression levels of phospho-ERK1/2 and
phospho-JNK after RA stimulation. Using the FACE ELISA
assay, the absorbance values obtained from the fetal smooth
muscle aorta, pulmonary artery, and arterial duct cells are
summarized in Figure 3A. The fetal arterial duct SMC
expressed the lowest level of phospho-ERK1/2 among these 3
cells. However, it increased significantly to 1.609±0.040 from
1.370±0.005 (absorbance: 450 nm, P<0.05, n=6). This action could not be cancelled by CD2366. The changes in the
pulmonary artery SMC did not reach significance after RA
treatment (Figure 3A).
The phospho-JNK was expressed differently. The SMC
from the arterial duct had the highest level of 0.371±0.006,
but the aorta had the level of 0.229±0.005 and the pulmonary
artery 0.239±0.006 (Figure 3B). After RA treatment, the
expression level in the arterial duct declined to 0.326±0.007
from 0.371±0.006 (P<0.05, n=6). Such an inhibition of
phospho-JNK expression could not be overcome by CD2366.
Discussion
The PDA often causes abnormal circulation of the blood
and harms children's health. Hypoxia may be involved in
this disease. If there are anatomic changes of the PDA in
adult patients, such as aortic aneurysm, calcification, or
being short and sometimes friable can further complicate
surgical treatment[17]. The closure of the arterial duct depends on
the smooth muscle contraction. Mutations in
the MYH11 gene affecting the C-terminal coiled-coil region of the smooth
muscle myosin heavy chain, a contractile protein of SMC,
could cause the disease[18]. The SMC appear to be different
in the arterial duct from other VSMC from the aorta and
pulmonary artery in our results. PCNA, which functions in DNA
replication and cellular proliferation processes, is expressed
significantly lower, while Bax and Bcl-2 are much higher in
arterial duct VSMC than those in aorta and pulmonary artery.
The protein of Bcl-2 is related to apoptosis and its activity is
regulated by a related 21 kDa protein known as Bax. An
increase in the ratio of Bax to Bcl-2 can lead to cell growth
inhibition and apoptosis[19]. A lower expression of PCNA
and higher expression of Bax indicate that arterial duct VSMC
grow in a slower rate than those from the aorta and
pulmonary artery.
Interestingly, RA stimulated the growth of arterial duct
VSMC, but not of aorta and pulmonary artery VSMC. After
arterial duct SMC exposure to RA, these cells proliferated,
as evidenced by the stimulation of PCNA expression and the
decreased Bax/Bcl-2 ratio. The proliferation-stimulated
effects of RA on the cells tested herein were generally reduced
by the co-administration of CD2366, an RAR(RA
receptor)-selective antagonist, which is an
adamantyl-methoxyphenyl-heptatrienoic acid
derivative[20]. This suggested that RA
stimulation of proliferation in the arterial duct was through
the RAR activation pathway. However, it was not clear
whether the regulation of Bax expression by RA was through
its receptors or not because CD2366 itself affects Bax
expression in the system. Additionally, in the arterial duct
SMC, high levels of phosphorylated ERK1/2 and low levels
of phosphorylated JNK were also noted. These results
demonstrated that RA induced proliferation in arterial
duct-derived VSMC, but not in aorta or pulmonary artery-derived
VSMC. Our results are consistent with a recent publication
in which the authors demonstrate that RA stimulates the
response of SMC in the ductus arteriosus to
platelet-derived growth factor and promote intimal cushion
formation[21]. Retinoid exposure during gestation can induce
developmental defects if administered before neurulation; however, if
given after neurulation, it is not so
teratogenic[22]. Exposure to idarubicin and RA early in the second trimester of
pregnancy causes transient dilated cardiomyopathy in a term
newborn[23]. However, some published studies have
suggested that vitamin A is important in the development of the
fetal ductus arteriosus[24,25]. Maternal administration of RA
may accelerate the mature process in the fetal ductus
arteriosus and the development of the
O2-sensing mechanism of the ductus
arteriosus[26]. We did not know the RA level in
the fetus blood because the blood was already clotted in our
fetus samples and therefore, could not be used for the
measurement. The content in the cultured VSMC was too
low to be detected by using high-pressure liquid
chroma-tography. However, evidence showed that endogenous RA
signaling is often colocalized with the expression of the adult
smooth muscle myosin heavy chain isoform during ductus
arteriosus development[27], which leads to the closure of the
ductus arteriosus. There is no evidence showing
prostaglandins specifically affect embryonic ducts rather than the
aorta or pulmonary artery, although prostaglandins were used
to induce the abortions.
The incidence of PDA has been associated with a
decreased mass of smooth muscles in the ductal wall, and RA
can induce proliferation of ductal SMC. Our findings
suggest that adequate RA may be useful for preventing PDA.
Conversely, excessive RA in the embryo can cause aortic
SMC to undergo apoptosis, such that an RA overdose may
lead to developmental defects of the aorta. Thus, improper
(either excessive or deficient) levels of retinoids may induce
teratogenesis during the development of the cardiovascular
system structures, such as the aorta, pulmonary artery, and
arterial duct.
We also observed changes in the arterial duct VSMC
from the embryos aged 16_20 weeks and noted that the
expression levels of PCNA and Bcl-2 were age related. This
illustrates that the proliferation level and anti-apoptotic
ability might decrease with increasing fetal age, although the
sample number and observation time points in this study
were limited. Whether or not this phenomenon is associated
with arterial duct occlusion after birth is worth additional
investigation. Whether the differences among the VSMC
from the arterial duct, aorta, or pulmonary artery are caused
by the different genesis of these VSMC or environment,
biology, or blood flow dynamics requires further
investigation using animal experiments.
Acknowledgements
We would like to thank Ms Xiao-hong GENG and Mr
Jian-bo WU for their help with the embryectomy.
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