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Original Studies |
INSERM U-361, Université René-Descartes, Maternité Baudelocque, 75014 Paris, France
Address all correspondence and requests for reprints to: Dr. C. Bourgeois, INSERM U-361, Maternité Baudelocque, 123 boulevard de Port-Royal, 75014 Paris, France. E-mail: u361{at}cochin.inserm.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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3, generated by
exclusion of exon 3 in stem villi vessels and PVSMCs. Alternative
splicing mechanisms of ETA-R mRNA could constitute a
control of the abundance of active ETA-R in terms of
contractility. PVSMCs will be a useful model to study the environmental
stimuli involved in the regulation of ET and ET-R expression in the
muscular layer of feto-placental vasculature. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ETs are a family of peptides (ET-1, ET-2, and ET-3) (for review, see 11 . Each of them is encoded by distinct genes from which peptidic precursors, the prepro-ETs (prepro-ET), are raised and subsequently cleaved to give the biologically active forms of ETs. Their receptors, ETA-R and ETB-R, belong to the superfamily of G protein-coupled receptors. The ETA subtype is selective for ET-1 and ET-2 compared to ET-3, whereas the ETB subtype is unable to distinguish among these three peptides.
In placental stem villi vessels, which are the major sites of placental vascular resistance, ET-1 exerts a direct vasoconstrictive effect on the smooth muscle layer of this vessels (8). To later investigate the influence of pregnancy-specific hormonal environment on the phenotype of the smooth muscle cells from these vessels and particularly on the regulation of ET and ET-R expression, we needed a model of isolated smooth muscle cells from stem villi vessels. In the present study, we aimed first to establish and characterize a culture of placental vascular smooth muscle cells (PVSMCs). Secondly, we investigated whether the muscular layer of stem villi vessels could itself be a site of ET production and examined this expression in PVSMCs. Finally, with the goal of using PVSMCs as ET target cells, we characterized the ET-R expressed by these cells compared with the tissue of origin, the smooth muscle layer of stem villi vessels from human term placenta.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human placentas were obtained aseptically, immediately after elective cesarean section, from healthy mothers in the 39th week of pregnancy. The cesarean sections, carried out before the onset of labor, were performed because of earlier diagnosed cephalopelvic disproportion.
Cell culture
Vascular smooth muscle cells were obtained by the explant method
with minor modifications of the technique described by Libby and
O’Brien (12). Briefly, immediately after elective cesarean section,
placental tissue (1–2 cm3) was swiftly removed from
between the decidual and chorionic plates and immersed in sterile
ice-cold Kreb’s solution (120 mmol/L NaCl, 5.7 mmol/L KCl, 2.5 mmol/L
CaCl2, 1.2 mmol/L NaH2PO4, 1.2
mmol/L MgCl2, 15.5 mmol/L NaHCO3, and 11.5
mmol/L glucose, pH 7.4). Branches of stem villi vessels (
200–300
µm of mean internal diameter) were dissected by fine scratching so
that the surrounding trophoblast, stroma, adventitia, and endothelial
cells were removed. They were then cut in pieces and thoroughly washed
under gentle magnetic agitation in 20 mmol/L Tris-HCl, pH 7.4,
containing 5 mmol/L L-leucine methyl ester (a
lysosomotropic compound that causes human monocyte death) (13) and used
for ribonucleic acid (RNA) extraction or for culture. Before placement
in culture, vessels were rinsed in DMEM supplemented with 10%
(vol/vol) FCS and 0.1% penicillin/streptomycin (Life Technologies,
Cergy-Ponroise, France). Finely cut pieces of placental vascular smooth
muscle (1–2 mm3) were then placed in 6-cm petri dishes
(Costar, D. Dutscher, France) and covered with one drop each of FCS.
The dishes were kept at 37 C in a humidified atmosphere of 5%
CO2-95% air. The following day, one drop of DMEM
containing 10% FCS was added to each explant. At 5 and 15 days of
culture, 2 mL DMEM containing 10% FCS were added to each dish. After
25–30 days, the dishes contained homogeneous layers of cells, which
were then subcultured. Cells were harvested with
trypsin-ethylenediaminotetraacetic acid, plated at 106
cells/75-cm2 flask, and grown to confluence in DMEM
supplemented with 10% FCS. Cells were studied from the second to the
sixth passage.
Cell cultures were tested by reverse transcription-PCR (RT-PCR) for the
expression of CD45 antigen messenger RNA (mRNA), a marker of
lympho-monocyte strain cells, and also for the expression of the von
Willebrand factor mRNA, a marker of endothelial cells (see Table 1
for oligonucleotide sequence of
primers).
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Cells grown to confluence in 24-well plates were rinsed twice
with phosphate-buffered saline (PBS) and fixed with 50%
ethanol-acetone (vol/vol) solution at -20 C for 3 min. After
rehydration in PBS for 20 min and blocking of nonspecific antigenic
sites by PBS containing 1.5% horse serum for 20 min, cells were
incubated overnight with a monoclonal antibody against smooth muscle
-actin, diluted at 1:20 in PBS containing 1.5% horse serum
(Novocastra, Newcastle, UK). Immunoreactions were detected using a
second biotinylated antibody and the
biotin/avidin-peroxidase/diaminobenzidine-Ni system (Elite ABC and
Vectastain kits, Vector, Biosys, France) according to the
manufacturer’s instructions.
RNA preparation and RT
Total RNA was extracted from vascular smooth muscle cells and stem villi vessels using a Quick II kit (Bioprobe, Montreuil, France). Total RNA from placental villi and trophoblast was obtained according to the method of Chomczynski and Sacchi (14). Human fibroblast (FS-4) RNA was prepared as previously described (15), and human lung RNA was obtained from Clontech (Ozyme, France). RT was performed as previously described (4) with 2 or 5 µg total RNA, using random hexanucleotides as primers and 200 U Moloney murine leukemia virus reverse transcriptase (Life Technologies) in a final volume of 25 µL at 37 C for 60 min.
PCR
The PCR reaction was performed as previously described (4).
Briefly, 5 or 10 µL of the RT reaction were subjected to
amplification in the presence of forward and reverse external
oligonucleotide primers (0.4 µmol/L each), deoxynucleotides (0.4
µmol/L each), and 1 U Taq polymerase (Life Technologies)
for typically 32 cycles in a DNA thermal cycler (PHC-3 Dri-Black
Cycler, Techné, Cambridge, UK). Forward and reverse external
oligonucleotides were designed to localize in separate exons and in the
least homologous regions (Table 1
). The specificity of each
amplification product was checked by Southern blot analysis as
previously described (4), except for the hybridization, which was
performed with an internal oligonucleotide labeled with
fluorescein-11-deoxy-UTP using an ECL 3' oligolabeling and detection
system kit (Amersham, France) according to the manufacturer’s
instructions.
Sequencing
After electrophoresis of the PCR product and ethidium bromide staining, the resulting DNA band was excised from the gel, treated with phenol-chloroform, and ethanol precipitated. The sequencing of the DNA template was performed in Genethon Laboratory (Evry, France) using the PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing Kit (Perkin-Elmer, Norwalk, CT).
Binding assay
Cell plasma membrane preparations and binding studies were performed as previously described (16). Briefly, aliquots of membranes (10 µg protein) were incubated for 60 min at 30 C in the presence of 5–300 pmol/L [125I]ET-1 (Amersham) for saturation analysis or in the presence of 50 pmol/L for competition studies. Specific binding of [125I]ET-1 was defined as total binding minus binding not displaced by 1 µmol/L unlabeled ET-1. At ligand concentrations near the dissociation constant (Kd), nonspecific binding was less than 30% of total binding for [125I]ET-1.
Competition experiments were carried out using a fixed concentration of [125I]ET-1 (50 pmol/L) with increasing concentrations of competing agents (Neosystem, Strasbourg, France).
All binding assays were performed at protein concentrations within the linear range. The inhibition constant (Ki) was calculated by the equation of Cheng and Prusoff (17), and Kd was determined by Scatchard analysis. Radioligand binding data were analyzed using the Inplot Computer program (GraphPad Software, San Diego, CA). The protein concentration was determined by the method of Lowry, using BSA as standard.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Morphology.
About 10 days after the explants of placental stem
villi vessels attached to the plate, PVSMCs began to migrate away from
the explants (Fig. 1A). PVSMCs formed a
continuous circular sheet around the explants after 3–4 weeks of
culture. Then, these cells were treated with trypsin and subcultured in
the presence of 10% FCS. They became confluent in 8–9 days. At
confluence, they were spindle shaped and exhibited a "hills and
valleys" appearance (Fig. 1B).
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-actin and smooth muscle myosin
heavy chains (MHCs).
Smooth muscle -actin: Immunocytochemical studies using an
antibody against smooth muscle -actin resulted in the staining of
long longitudinal filaments throughout the cytoplasm of confluent
PVSMCs (Fig. 1C). No staining was obtained when the same cells were
incubated with the secondary antibody alone, thus assessing the
specificity of the labeling (Fig. 1D).
MHCs: Smooth muscle MHC isoforms (SM1 and SM2) arise by
alternative RNA splicing from a single gene, SM-MHC. Inclusion of a
39-nucleotide stretch generates SM2, and its exclusion produces SM1
(27). The expression of smooth muscle MHC isoform mRNAs was
investigated in confluent PVSMCs and placental stem villi vessels by
RT-PCR, using external oligonucleotides specific for SM1 and SM2 mRNA
sequences. When the resulting complementary DNA (cDNA) fragments were
analyzed by electrophoresis and ethidium bromide staining of the
agarose gel, two products of the predicted size (426 bp for SM2 and 387
bp for SM1) were obtained in placental stem villi vessels (Fig. 2, lane 3), whereas only the 387-bp
fragment could be detected in PVSMCs (Fig. 2, lane 1). No amplification
product was observed when reverse transcriptase was omitted (Fig. 2, lane 2). However, after Southern blotting of RT-PCR products and
hybridization with an SM2-specific internal oligonucleotide, a cDNA
fragment (426 bp) corresponding to SM2 was labeled in PVSMCs (Fig. 2, lane 4) as well as in placental stem villi vessels (Fig. 2, lane 5). No
amplification product was observed in human fibroblasts (Fig. 2, lane
6). In PVSMCs, we also checked the absence of von Willebrand factor and
CD45 antigen expression, which are markers of endothelial and
lympho-monocyte strain cells, respectively (data not shown).
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Prepro-ET-1.
The expression of the ET-1 gene was investigated
in total RNA prepared from confluent PVSMCs, stem villi vessels, and
human lung, used as a positive control. After RT-PCR amplification
using external oligonucleotides specific for the prepro-ET-1 sequence,
a product of the predicted size (442 bp) was obtained in the positive
control human lung (Fig. 3A, lane 1), in
stem villi vessels (Fig. 3A, lane 2), and in PVSMCs (Fig. 3A, lane 4),
as revealed by ethidium bromide staining after agarose gel
electrophoresis. No amplification product was observed when reverse
transcriptase was omitted (Fig. 3A, lane 3). After Southern blotting
and hybridization with an internal prepro-ET-1-specific
oligonucleotide, the 442-bp amplification products from human lung
(Fig. 3A, lane 5), stem villi vessels (Fig. 3A, lane 6), and PVSMCs
(Fig. 3A, lane 7) were labeled.
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, lane 3), whereas no
amplification product could be detected in PVSMCs (Fig. 3B, lane 1) or
stem villi vessel mRNAs (Fig. 3B, lane 2).
Prepro-ET-3.
The expression of prepro-ET-3 mRNA was
investigated in the same stem villi vessels and PVSMC cDNAs. After
Southern blotting of RT-PCR products and hybridization with an internal
prepro-ET-3-specific oligonucleotide, an amplification product of the
predicted size (479 bp) was labeled in the human trophoblast used as a
positive control (Fig. 3C, lane 1) and in stem villi vessels (Fig. 3C, lane 3). In contrast, no amplification could be detected in PVSMCs
(Fig. 3C, lane 2). When reverse transcriptase was omitted, no
amplification product was observed (Fig. 3C, lane 4).
mRNA expression of ET-R
In humans, two distinct genes encoding for ETA-R and ETB-R have been described to date. The expression of ETA-R and ETB-R subtype mRNAs was investigated by RT-PCR in confluent PVSMCs and in stem villi vessels.
ETA-R.
Using external
ETA-specific oligonucleotides (Fig. 4A, PCR 1), an amplification product of
the predicted size (626 bp) and two shorter fragments (498 and 299 bp)
were obtained in human placental villi, which was used as a positive
control (Fig. 4B, lane 1), in PVSMCs (Fig. 4B, lane 2), and in stem
villi vessels (Fig. 4B, lane 4) as revealed after ethidium bromide
staining of agarose gel. No amplification product was observed when
reverse transcriptase was omitted (Fig. 4B, lanes 3 and 5). After
Southern blotting and hybridization with an internal oligonucleotide
(208–227) specific for the sequence of ETA-R gene exon 2,
the 626-bp fragment as well as the 498- and 299-bp fragments were
labeled in the three samples (Fig. 4B, lanes 6–8). In addition, a band
corresponding to a 427-bp product was labeled using ETA
exon-2 probe in human placental villi (Fig. 4B, lane 6) and stem villi
vessels (Fig. 4B, lane 8), whereas it was not observed in PVSMCs (Fig. 4B, lane 7). The 427- and 299-bp PCR products correspond respectively
to ETA-R4 and ETA-R3,4 mRNAs, two
transcripts previously described in the human placenta (27). In
contrast, the 498-bp fragment did not correspond to any previously
described ETA-R mRNA-spliced fragment. The difference in
size between the 626-bp product of the full-length ETA-R
transcript and the newly described 498-bp fragment (Fig. 4B) is
consistent with exclusion of exon 3 alone. To verify this hypothesis,
we performed another amplification using the same forward external
oligonucleotide but with an exon 4-specific primer as the reverse
external oligonucleotide (Fig. 4A, PCR 2). After hybridization with
exon 2-specific probe, two amplification products of the expected sizes
(556 and 428 bp, respectively) were labeled in PVSMCs (Fig. 4C, lane 1)
and stem villi vessels (Fig. 4C, lane 3) as well as in human placental
villi (data not shown). No amplification product was observed when
reverse transcriptase was omitted (Fig. 4C, lanes 2 and 4).
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. The splicing pattern (Fig. 5B) will
result in a shift in the open reading frame of the ribonucleic sequence
position and in the reading of a stop codon at the 22 amino acid
position in exon 4 (Fig. 5A).
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, lane 2), whereas no labeling was
observed in PVSMC (Fig. 6, lane 4). No amplification product was
observed when reverse transcriptase was omitted (Fig. 6, lanes 1 and
3).
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In PVSMCs, the addition of increasing concentrations (5–300
pmol/L) of [125I]ET-1 resulted in specific and saturable
binding (Fig. 7A, inset).
Scatchard analysis of the saturation curve indicated the presence of a
single class of high affinity binding sites with an apparent
dissociation constant (Kd) of 52 ± 6 pmol/L and a
maximal capacity of 54.5 ± 6.5 fmol/mg protein (n = 4; Fig. 7A). The calculated number of receptors was 6426 ± 599
sites/cell. As shown in Fig. 7B, the binding of
[125I]ET-1 was competitively inhibited by ET-1 with a
Ki value of 1.7 ± 0.5 nmol/L, whereas a selective
ETB-R agonist, sarafotoxin 6c (S6c), was inactive up to 1
µmol/L. A selective ETA-R antagonist, BQ-123, caused
total displacement in an apparent monophasic fashion with a
Ki value of 2.2 ± 0.3 nmol/L. In contrast, the
ETB antagonist IRL 1038 failed to displace
[125I]ET-1 binding (Fig. 7C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3, generated by exclusion of exon 3 alone.
Due to extensive modulation of their phenotype between the contractile
and the proliferative states, cultured smooth muscle cells (for review,
see 29 are notoriously difficult to characterize. A number of
structural and regulatory proteins associated with smooth muscle cells
allow their discrimination from fibroblasts, macrophages, and
endothelial cells. PVSMCs obtained from explants of the stem villi
vessel muscular layer collected from human term placenta exhibit the
characteristics of explant-derived vascular smooth muscle cells: they
grow to confluence with a hills and valleys pattern and express smooth
muscle -actin, a contractile protein that appears to be the earliest
marker of differentiated smooth muscle. We also sought the expression
of smooth muscle myosin heavy chain isoforms, SM1 and SM2, which are
considered to be the most rigorous known markers for identification of
differentiated smooth muscle cells. Although SM1 and SM2 protein
expression was previously reported in stem villi vessels (30), these
proteins were barely detectable in PVSMCs when using immunological
tools. However, by RT-PCR we demonstrated the presence of SM1 and SM2
transcripts in PVSMCs, whereas these transcripts were not detected in
fibroblasts. According to these morphological (hills and valleys
pattern) and biochemical (contractile proteins) criteria, we concluded
that PVSMCs were of smooth muscle lineage. As these cells were intended
to be used to study the expression of ET and their receptors by RT-PCR,
a very sensitive method, we also checked by this method the absence of
contaminating cells able to synthesize ET such as endothelial and
lympho-monocyte strain cells. Only the PVSMC RNA preparations found to
be negative for the expression of these contaminating cell markers were
selected to perform the following studies.
We showed the expression of prepro-ET-1 and prepro-ET-3 transcripts in the muscular layer of placental stem villi vessels, whereas we identified only prepro-ET-1 mRNAs in cultured PVSMCs (second to sixth passage). In contrast, prepro-ET-2 mRNAs were not expressed in placental stem villi vessels or in PVSMCs. In the conditioned medium of subcultured PVSMCs (second to third passage), preliminary results indicate a weak production of immunoreactive ET-1 (order of magnitude, 12 fmol/106 cells·24 h) (Bourgeois, C., et al., unpublished data). Our present results suggest that in the feto-placental vasculature, the smooth muscle cell itself is involved in the production of a vasoactive substance that can modulate its contractile activity in an autocrine/paracrine way.
With the aim of using PVSMCs as ET target cells, we characterized the ET-R expressed by these cells compared with the smooth muscle layer of stem villi vessels from human term placenta. In stem villi vessels, we showed the expression of ETA-R and ETB-R subtype mRNAs. These results are in agreement with our previous report describing the presence of ETA and ETB binding sites coupled to the phospholipase C/Ca2+ signaling pathway (31). In contrast, in PVSMCs only the ETA-R mRNAs were identified. Using agonists and antagonists selective for the ET-R subtypes, we confirmed the exclusive presence of ET-binding sites of the ETA subtype on PVSMCs. Their Kd value (52 ± 6 pmol/L) is consistent with Kd values previously reported in stem villi vessels (26 ± 4 pmol/L) (9).
Compared to their tissue counterparts, prepro-ET-3 and ETB-R expression were not found in PVSMCs. This alteration of gene expression pattern could occur in response to placement in culture. Subculturing has also been associated with the disappearance of ETB-R expression in tracheal smooth muscle cells (32) and in myometrial cells (16). This is not surprising given the plasticity of smooth muscle cells and the fact that it is nearly impossible to perfectly mimic in culture the in vivo conditions. Alternatively, the prepro-ET-3 and ETB-R expression observed in the smooth muscle wall of stem villi vessels could be due to the presence of contaminating cells and, consequently, would not be observed in isolated PVSMCs.
In addition to the full size ETA-R mRNA, we found that the
ETA-R gene gives rise to alternatively spliced
ETA-R transcripts in stem villi vessels as well as in
PVSMCs. Two of them, respectively called ETA-R4 and
ETA-R3,4, were recently described in placenta and other
human tissues (28). In addition, we report in stem villi vessels and
PVSMCs, the expression of a third ETA-R mRNA,
ETA-R3, in which exon 3 alone is excluded. ET-R belong
to the group of G protein-coupled receptors that are characterized by
their seven-membrane-spanning domain structure (for review, see 33 . In the ETA-R3 transcript, exclusion of exon 3
results in the reading of a stop codon at the beginning of exon 4.
Consequently, translation of ETA-R3 transcripts should
generate a very short ETA-R form with only
membrane-spanning domains I and II according to their putative location
(21). Similarly, translation of ETA-R3,4 and
ETA-R4 mRNAs should generate very truncated
ETA-R proteins with only five and three membrane-spanning
domains, respectively (28). Interestingly, a similar splicing mechanism
leading to truncated transcripts has been described for another G
protein-coupled receptor, the dopamine receptor D3
(34).
Despite the presence of several ETA-R mRNAs, we identified
a single class of ETA-binding sites on PVSMCs. This could
be due to the fact that pharmacological studies produce rather global
results that do not always allow one to distinguish between subtypes.
However, given the truncated structure of ETA-R3 and
4, it is unlikely that these proteins function as receptors. In the
case of ETA-R3,4, for which five membrane-spanning
domains would be conserved, Miyamoto et al. (28) reported
that COS-7 cells transiently transfected with ETA-R3,4
cDNA did not exhibit any specific binding affinity for
[125I]ET-1 on their plasma membrane. These data are
consistent with chimera ET-R results, which suggest that all of the
membrane-spanning domains were involved in ligand binding to
ETA-R (35). The splicing mechanism of ETA-R
mRNA may constitute a posttranscriptional control of the abundance of
active ETA-R. In stem villi vessels, ETA-R
activation is involved in the constrictive effect of ET-1. Therefore,
at the end of pregnancy characterized by a low placental vascular
resistance, the generation of truncated ETA-R transcripts
in stem villi vessels may constitute a mechanism controlling the amount
of functional ETA-R involved in contraction and possibly in
proliferation/differentiation processes.
In conclusion, we developed a cell culture model of smooth muscle cells from placental stem villi vessels in which we report the expression of ET-1 mRNA and the presence of ETA-R. The splicing mechanism by which multiple ETA-R transcripts arise in stem villi vessels is maintained in PVSMCs. Throughout pregnancy, the placenta has to provide for fetal nutritional needs, especially near term, when the needs drastically increase. In response to this demand, placental vascularization undergoes modifications of tone and probably structural features. Little is known of the mechanisms responsible for the physiological adaptation of the feto-placental vasculature during pregnancy. PVSMCs in culture will constitute a useful cell model to study the impact of pregnancy-specific stimuli, such as steroid hormones, on the regulation of ETs and ET-R expression in the smooth muscle layer of feto-placental vasculature.
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Acknowledgments |
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Footnotes |
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3-R cDNA reported in
this paper has been submitted to the GenBank/EMB Data Bank with
accession number: AF014826. 
Received September 10, 1997.
Revised March 17, 1997.
Accepted May 28, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-like mRNAs and high expression of interleukin-6 in
human fetal annexes. J Biol Chem. 264:16507–16511.This article has been cited by other articles:
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