© 1996 American Heart Association, Inc.
Articles |
Regulation of Endothelial Production of C-Type Natriuretic Peptide in Coculture With Vascular Smooth Muscle Cells
Role of the Vascular Natriuretic Peptide System in Vascular Growth Inhibition
From the Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Japan.
Correspondence to Hiroshi Itoh, MD, PhD, Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606, Japan.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults Discussion References |
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Abstract Recently, we have demonstrated that C-type natriuretic peptide (CNP) is produced in vascular endothelial cells (ECs). In the present study, we investigated the interaction of ECs and vascular smooth muscle cells (SMCs) for endothelial production of CNP and its action on vascular growth, using the EC/SMC coculture system. The concentration of CNP-like immunoreactivity in the medium was increased 60-fold within 48 hours in the EC/SMC coculture with direct contact compared with that in EC alone. Northern blot analysis revealed the augmented expression of CNP mRNA in the EC/SMC coculture. The accumulation of intracellular cGMP in the coculture was concomitantly increased, and this response was blocked by anti-CNP monoclonal antibody and HS-142-1, a nonpeptide atrial natriuretic peptide receptor antagonist. The concentration of biologically active transforming growth factor-ß (TGF-ß) in the culture medium of the coculture with direct contact of ECs and SMCs was elevated to the level to stimulate endothelial production of CNP. Actually, the neutralizing antibody against TGF-ß abrogated the cGMP accumulation in the coculture. These results show that endothelial production of CNP in the EC/SMC coculture is at least in part regulated by TGF-ß. Furthermore, the conditioned medium from ECs stimulated by TGF-ß was demonstrated to have a growth-inhibitory effect on SMCs, which was abolished by anti-CNP monoclonal antibody and HS-142-1. The treatment with anti-CNP monoclonal antibody and HS-142-1 also significantly increased the cell number of the EC/SMC coculture. The present study reveals the pathophysiological significance of endothelial CNP as a paracrine/autocrine vascular regulator for vascular growth in the interaction of ECs and SMCs.
Key Words: cyclic GMP • natriuretic peptide receptor • endothelium-derived relaxing factor • transforming growth factor-ß
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults Discussion References |
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Natriuretic peptides comprise a family of three distinct peptides: ANP, BNP, and CNP and are involved in body fluid homeostasis and blood pressure control. CNP, the third member of the natriuretic peptide family, was originally isolated from the porcine brain. It has 22 amino acid residues and shows a sequence homology to ANP and BNP within the ring structure formed by an intramolecular disulfide linkage.1 Although we showed that the major production sites for ANP and BNP are the atrium and ventricle of the heart, respectively,2 CNP is thus far reported to be distributed not in the heart but mainly in the central nervous system in rats and humans.3
The ANP-A and ANP-B receptors are the particulate guanylate cyclase and elicit an increase in intracellular cGMP that mediates most of the biological actions of the natriuretic peptides, whereas the clearance receptor is thought to be involved in clearance of the natriuretic peptides.4 We and others investigated the ligand receptor relationship of the natriuretic peptide system and demonstrated that the ANP-B receptor is selectively activated by CNP.5 6 The ANP-B receptor has been shown to occur not only in the brain but also in the peripheral tissues, including blood vessels.7 8 The peripheral presence of the ANP-B receptor and the systemic action of CNP9 10 made us speculate that CNP also exists and acts as a local regulator in the peripheral tissues.
In this context, we recently discovered that cultured ECs produce
CNP11 and demonstrated the transcripts of CNP and ANP-B
receptor genes simultaneously in human vascular walls in
vivo.7 We and others also demonstrated that ANP and CNP
possess a vascular growth-inhibitory
action.12 13 Furthermore, we recently found that
TGF-ß,
one of the potent growth factors involved in vascular growth, quite
potently increases endothelial production of
CNP.11 In addition, we observed that various kinds of
cytokines, especially tumor necrosis factor-
, augment CNP
production as much as TGF-ß.14 More recently, we
further demonstrated the existence of CNP in human plasma and the
marked increase of the plasma CNP level in septic shock
patients.10 15 Therefore, we have proposed the
existence
of a vascular natriuretic peptide system, regulating
vascular tone and growth.
Vascular remodeling plays a pivotal role in the pathogenesis of many cardiovascular disorders, especially hypertension and atherosclerosis. Proliferative vascular lesions occur in the complicated interaction between ECs and SMCs. To elucidate the significance of the vascular natriuretic peptide system in vascular remodeling, in the present study we have used the coculture technique16 to define the consequence of the interaction of ECs and SMCs. We have also explored the molecular mechanism of the augmentation of CNP production and further examined the function of endothelial CNP in the growth of SMCs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults Discussion References |
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Cell Cultures
ECs were isolated from the initial lining of adult bovine carotid arteries.11 The cells were suspended in DMEM (Flow Laboratories, Inc) containing 10% FCS (Hazleton Biologics, Inc). Cultures were incubated at 37°C in 95% air/5% CO2 and were passaged after treatment with trypsin (0.25%, GIBCO). Cells were verified as ECs by their phase microscopic "cobblestone" appearance and their ability to take up acetylated low-density lipoprotein. Experiments were performed by using cells of passages 12 through 20. Cultured SMCs were obtained from the explants of thoracic aortas of male Wistar rats and cultured as described previously.8 Cells in subculture passages 11 through 15 were used for all experiments, since progressive decline of the activity of cGMP-dependent protein kinase, which is one of the major mediators for cGMP actions, in later cell passages is reported.17 L6 cells (rat skeletal muscle myoblasts)18 were given by Japanese Cancer Research Resources Bank,Tokyo.
Coculture Studies
Passaged ECs and SMCs were cocultivated.
Briefly, ECs were grown
to confluence on 24-well tissue-culture dishes. The indicated
number of SMCs was plated over the confluent monolayer of ECs,
containing 2x105 cells. After overnight attachment, cells
were washed with PBS twice, given 250 µL of DMEM containing 0.5%
FCS, and incubated for the indicated time.
Additional coculture experiments were conducted using Millicell chambers (Millipore Corp) to determine the role of cell contact. In this system, the cells were cocultured in the same well, but physical interaction was prevented by growing SMCs in the Millicell chamber. All other experimental conditions were identical.
Radioimmunoassay for CNP
The radioimmunoassay for CNP was
performed with mouse CNP
polyclonal antisera, which we developed.3 The
cross-reactivities with -ANP, porcine BNP, rat BNP, and CNP-53
were 0.2%, 14%, <0.01%, and 100% on a molar basis, respectively.
The cross-reactivities with ET-1, -2, and -3 were <0.01% on a
molar basis. Measurement of ET-1 concentrations was performed as
described.19
RNA Extraction and Northern Blot Analysis
RNA was extracted
from cells by the guanidinium thiocyanate
cesium chloride method and was subjected to poly(A)+ RNA
enrichment. RNA was electrophoresed on a formamide/1.2% agarose gel
and transferred to a nylon membrane filter. The CNP cDNA probe (381
bp), including the entire coding region for human CNP, was
prepared.11 The human G3PDH cDNA probe was purchased from
Clontech Inc (No. 9205-1).20 The filters were hybridized
with a 32P-labeled probe at 42°C in 50% formamide, 5x
SSC, 5x Denhardt's reagent, 50 mmol/L sodium phosphate buffer (pH
6.8), 0.l% SDS, and 100 mg/L heat-denatured salmon sperm DNA,
washed at 65°C in 0.1x SSC and 0.1% SDS.11
Autoradiography was performed on x-ray films
with intensifying screens, and the relative amount of each mRNA was
determined by densitometric scanning in the linear response range of
the x-ray films.
Intracellular cGMP Determination
ECs, SMCs, and the mixed
population were given 250 µL of DMEM
containing 0.5% FCS. After a 48-hour incubation of the coculture,
isobutylmethylxanthine (Sigma Chemical Co) was
added (0.5 mmol/L). The cells were incubated for an additional 30
minutes at 37°C. The medium was then rapidly removed and 250 µL of
ice-cold 6% trichloroacetic acid was added to the dish. The cell
samples were centrifuged to remove precipitated proteins, and
the supernatant fractions were extracted three times with
water-saturated ether. The concentration of cGMP was determined by
radioimmunoassay after succinylation, as previously
described.6 Protein content was measured by the Bradford
method.
Effects of Monoclonal Anti-CNP Antibody and HS-142-1
Anti-CNP
monoclonal antibody (KY-CNP-I) was prepared against
synthetic CNP.21 Purified IgG was prepared by protein A
chromatography. The neutralizing effect of KY-CNP-I was
confirmed on cGMP production of cultured SMCs in response to
CNP. The preincubation with 3 mg/L KY-CNP-I resulted in an almost
complete inhibition of cGMP accumulation in SMCs induced by 10 nmol/L
CNP. The nonpeptide natriuretic peptide receptor
antagonist HS-142-1 was a generous gift of Y. Matsuda at
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co, Ltd,
Tokyo.22 KY-CNP-I (3 mg/L), nonimmune mouse IgG (3 mg/L)
(Sigma Chemical Co), or HS-142-1 (1, 10, or 100 mg/L) was added to the
coculture dishes after SMC attachment. After 48 hours' incubation, the
cells were subjected to determination of the cGMP concentration.
Bioassay for TGF-ß and Neutralization of TGF-ß
Action
The level of TGF-ß in the conditioned medium was determined
by
bioassay, using CCL-64 mink lung epithelial cells as
described.23 The concentrations of TGF-ß in the
conditioned medium obtained from ECs alone, SMCs alone, and the EC/SMC
coculture in a 48-hour period were determined by the standard curve
prepared with various concentrations of TGF-ß (human TGF-ß, R&D
Systems). The minimal detectable quantity in this assay was 5.0 pmol/L.
The specificity of this assay was confirmed by the abolishment of the
growth-inhibitory activity by the prior incubation of
the conditioned medium with the specific TGF-ß neutralizing antibody
(described below).24 The concentrations of total TGF-ß
were estimated by acid activation of the conditioned medium as
previously described.25
Rabbit anti-porcine TGF-ß IgG was purchased from R&D Systems.26 This antibody has been shown to neutralize both porcine and human TGF-ß. The dosage that produces 50% neutralization was determined to be 3 to 5 mg/L antibody in neutralization of 10 pmol/L TGF-ß. Twenty milligrams per liter of either anti–TGF-ß IgG or nonimmune rabbit IgG (Zymed Laboratories, Inc) was added to the coculture dish. After a 48-hour incubation, the cells were subjected to the determination of cGMP concentration.
Evaluation of SMC Growth-Inhibitory Property of
Endogenous CNP from ECs
The medium conditioned with ECs was prepared
and investigated
for the action on DNA synthesis of SMCs. The confluent culture of ECs
was washed twice with PBS and given DMEM containing 0.5% FCS and 100
pmol/L TGF-ß. After a 24-hour incubation, the conditioned medium was
collected under sterile conditions and assayed or subjected to further
treatment.
Relative rates of DNA synthesis of SMCs were assessed by determination of [3H]thymidine incorporation into trichloroacetic acid–precipitable material. In preparation for experiments, SMCs were made quiescent by placing them for 48 hours in a defined serum-free medium containing insulin, transferrin, and ascorbate (Sigma Chemical Co).12 The cells were washed once and given DMEM containing 0.5% FCS or EC-conditioned medium. During the incubation, CNP (10 nmol/L), control mouse IgG (15 mg/L), KY-CNP-I (15 mg/L), or HS-142-1 (10 mg/L) was also added. After 48 hours' incubation, they were subjected to [3H]thymidine incorporation assay.12
In another series of experiments, the EC/SMC coculture was established, with an equal number of ECs and SMCs (1x105 cells) in DMEM containing 0.5% FCS. KY-CNP-I (15 mg/L), control mouse IgG (15 mg/L), or HS-142-1 (10 mg/L) was added, and after 48 hours' incubation, the cells were harvested to determine the cell number as described previously.12
Data Analysis
Data are presented as mean±SEM of the
indicated number
of individual cultures. In our experiments, n=4 or 6 (two independent
samples in two or three separate experiments). Statistical comparisons
between groups were performed using one-way ANOVA followed by the
Newman-Keuls multiple-range test, unless indicated otherwise.
Differences among means were considered significant at a value of
P<.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResults Discussion References |
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CNP Secretion From ECs in EC/SMC Coculture
We have examined the effect of the EC/SMC coculture on endothelial production of CNP. Fig 1
depicts the time course of CNP-LI accumulation in the
culture-conditioned medium. CNP-LI was detected (46.1±2.2 pmol/L)
in the conditioned medium of ECs alone at 48 hours (dashed line in Fig
1). In contrast to ECs, we could not detect CNP-LI (<4.0
pmol/L) in
the conditioned medium of SMCs. The concentration of CNP-LI in the
EC/SMC coculture medium was dramatically elevated and reached up to
2.68±0.47 nmol/L at 48 hours (solid line in Fig 1). The
EC/SMC
coculture induced a 60-fold increase of CNP-LI concentration in the
conditioned medium compared with ECs alone. In contrast, the
concentration of ET-l–like immunoreactivity in the EC/SMC
coculture medium at 48 hours (6.02±0.57 nmol/L) was 1.3-fold higher
than that in the medium of ECs alone (4.57±0.31 nmol/L). The
stimulating effect of the EC/SMC coculture, therefore, seems to be much
more potent and favorable for CNP secretion than ET secretion. Neither
ANP-like immunoreactivity nor BNP-like immunoreactivity was detected in
the conditioned medium and cell extracts from the EC/SMC coculture
(<0.4 pmol/L) at 48 hours.
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To assess the influence of the ratio of
SMCs to ECs on
endothelial production of CNP, variable
numbers of SMCs (5%, 10%, 20%, 50%, and 100% of EC number) were
plated on a confluent monolayer of ECs (2x105 cells per
well). The coculture of ECs with SMCs augmented the accumulation of
CNP-LI in the conditioned medium at 24 hours in an SMC
number–dependent manner (20% to 100%) (Fig 2).
However, when ECs were cocultured with a lower number of SMCs (5% or
10% of EC number), the concentration of CNP-LI was not different from
that of ECs alone.
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The specificity of the augmentation of endothelial
production of CNP with SMCs was assessed by establishing the
coculture of ECs with the same number of L6 cells (2x105
cells per well). No significant elevation of
endothelial production of CNP was observed in
the EC/L6 coculture (Fig 2
).
When contact or proximity between ECs and SMCs was prevented by using Millicell chambers, no enhancement of endothelial production of CNP was observed in the coculture (24.1±1.5 pmol/L in the EC/SMC coculture with Millicell chambers versus 22.5±1.2 pmol/L in ECs alone after 24 hours' incubation).
Fig
3 depicts the time course of CNP-LI accumulation in
the conditioned medium of the coculture with and without direct
contact. The SMC number was 25% of the EC number in this experiment.
The CNP concentration in the culture medium was 228±27 pmol/L after 48
hours' incubation in the coculture with direct contact. In contrast,
the CNP concentration in the EC/SMC coculture without direct contact
was 45±8 pmol/L, which was not significantly different from that in
the EC culture alone.
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indicates ECs alone; 
,
coculture of ECs and SMCs without direct contact (SMCs were grown in
Millicell chambers); 
, coculture of ECs and SMCs with direct
contact. The cell numbers of ECs and SMCs were 2x105 and
0.5x105 per well, respectively. Each point
represents mean±SEM (n=4). *P<.05 vs ECs
alone.
In another series of experiments, we examined the
cell polarity of
endothelial cells, the importance of which is suggested
by several reports.27 We investigated CNP secretion by
using a coculture system in which SMCs were first seeded and ECs of
equal number were placed over them to grow. CNP-LI in coculture of ECs
over SMCs and EC culture alone were 2.2±0.12 nmol/L and 40±15
pmol/L
(n=4), respectively, after 48 hours' incubation. These values
were
comparable to those in the coculture of SMCs over ECs (Fig 1).
Regulation of Endothelial CNP mRNA Expression in
EC/SMC Coculture
Northern blot analysis detected CNP mRNA with a size
of 1.2 kb in a limited quantity in ECs alone (Fig 4, lane
1), as we previously reported.11 In the
EC/SMC coculture for 12 hours, two species of CNP mRNA with the size of
1.2 and 0.9 kb were expressed at intense level (11-fold versus ECs
alone) (lane 5). The intensity of the hybridizing bands for CNP mRNA in
the cocultured cells was comparable to that in ECs stimulated by
TGF-ß (100 pmol/L) for 12 hours (lane 2), while a weak band was
detectable in either SMCs alone or SMCs exposed to TGF-ß (100 pmol/L)
(lanes 3 and 4, respectively). G3PDH mRNA expressions were not
different in these lanes.
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cGMP Production in EC/SMC Coculture and Effects of
Neutralizing Antibody Against CNP and HS-142-1
When ECs and SMCs were
cocultured for 48 hours, the basal
intracellular cGMP level in the EC/SMC coculture was dramatically
elevated above the levels in SMCs alone and ECs alone (Fig 5A,
open bars).
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Incubation of the EC/SMC coculture with 3 mg/L KY-CNP-I
virtually
abolished the elevation of cGMP in the coculture, while control mouse
IgG had no significant effect on cGMP levels (Fig 5A). Neither
treatment affected basal cGMP production in either ECs alone or
SMCs alone (Fig 5A).
Incubation of the EC/SMC coculture
with HS-142-1 inhibited the
elevation of intracellular cGMP production in the coculture in
a concentration-dependent manner (1, 10, or 100 mg/L), while basal
cGMP production of either ECs alone or SMCs alone did not
change with HS-142-1 (Fig 5B).
TGF-ß Activity in EC/SMC Coculture and Neutralization of
TGF-ß Action
TGF-ß is one of the most potent stimulators of
endothelial production of CNP11
and has been demonstrated to be released by SMCs and/or
ECs.26 Therefore, we investigated the possibility that
TGF-ß is the active component of the conditioned medium of the EC/SMC
coculture for the stimulation of CNP production. The dilution
curve of the conditioned medium obtained from the EC/SMC coculture was
parallel to the standard curve of TGF-ß in the bioassay using CCL-64
cells. The conditioned medium from ECs alone did not affect the DNA
synthesis of CCL-64 cells even after the acid activation. The level of
active TGF-ß in the conditioned medium from the EC/SMC coculture (the
numbers of ECs and SMCs were 2x105 cells per well) was
five times higher than that from SMCs alone. Total TGF-ß
concentrations were similar between SMCs alone and the EC/SMC coculture
(Table).
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Coincubation with 20 mg/L of the neutralizing
antibody against TGF-ß
significantly reduced intracellular cGMP accumulation in the EC/SMC
coculture compared with the basal level, although nonimmune IgG had no
significant effect (Fig 6). The neutralizing
anti–TGF-ß antibody elicited no significant effect on the cGMP
levels in ECs alone or SMCs alone.
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Furthermore, we compared active
TGF-ß concentration in the EC/SMC
coculture with versus without direct contact, as described above (Fig
3). Active TGF-ß level in the EC/SMC coculture with direct
contact
after 48 hours' incubation was 20.6±1.1 pmol/L (n=4),
while that in
the EC/SMC coculture without direct contact was <5 pmol/L (n=4).
Inhibitory Action of Endothelial CNP on
SMC Growth
Since the EC/SMC coculture contains a mixed cell
population, it is
difficult to examine the growth of SMCs themselves. Therefore, the
conditioned medium from ECs was prepared to examine the effect of
endothelial CNP on SMC growth. When ECs were stimulated
with TGF-ß at the dose of 100 pmol/L, the concentration of CNP-LI in
the conditioned medium was 2.90±0.16 nmol/L at 24 hours, which is
similar to that in the EC/SMC coculture (2.68±0.47 nmol/L).
SMCs
were made quiescent by incubating them with defined serum-free
medium for 48 hours. Cells cultured in this manner possess a low basal
level of [3H]thymidine incorporation (18 500±2650
cpm
per well), which is actively stimulated by exposure to DMEM containing
0.5% FCS (79 400±7800 cpm per well; control in Fig 7).
[3H]Thymidine incorporation was
inhibited by 38% by 10 nmol/L CNP. This antiproliferative action of
CNP was abolished by KY-CNP-I (15 mg/L) and HS-142-1 (10 mg/L), while
control IgG (15 mg/L) had no significant effect (Fig 7). In
this
setting of the experiment, when SMCs were treated with EC-CM,
[3H]thymidine incorporation was similar to that in the
control. Treatment with KY-CNP-I (15 mg/L) and HS-142-1 (10 mg/L)
significantly enhanced the [3H]thymidine incorporation by
40%, while control IgG (15 mg/L) had no significant effect (Fig
7).
Neither treatment gave any change of [3H]thymidine
incorporation in the control. In another series of experiments, it was
confirmed that treatment with EC-CM enhanced the intracellular cGMP
accumulation of SMCs and that the increase of cGMP accumulation was
completely abolished by KY-CNP-I and HS-142-1.
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P<.05 vs
control.Furthermore, we examined SMC proliferation in the coculture after the blockade of endogenous CNP from ECs. Treatment with KY-CNP-I (15 mg/L) caused significant increase of the cell number in the EC/SMC coculture (6.4±0.3x105 per well) compared with the control IgG–treated group (4.7±0.3x105 per well, P<.05). The administration of HS-142-1 (10 mg/L) also significantly increased the cell number in the coculture (6.5±0.3x105 per well) compared with the vehicle-treated group (5.4±0.2x105 per well, P<.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResults Discussion References |
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In the present study, using the EC/SMC coculture system, we demonstrated that the interaction of ECs and SMCs markedly augments endothelial production of CNP. This augmentation is profound (60-fold) compared with the effect on ET secretion (1.3-fold). Since the increase of CNP production in the coculture depends on the number of SMCs, and neither the coculture of ECs with L6 cells (rat skeletal myoblasts) nor the coculture using Millicell chambers had significant effect, the stimulating effect on endothelial production of CNP in the EC/SMC coculture is the specific consequence of the direct interaction of ECs and SMCs.
The present study also showed that intracellular cGMP accumulation in the EC/SMC coculture increased within 48 hours after the initiation of the coculture and that the increased accumulation of cGMP in the EC/SMC coculture was attenuated by the anti-CNP monoclonal neutralizing antibody KY-CNP-I and the nonpeptide natriuretic peptide receptor antagonist HS-142-1. The observed increase of cGMP production in the EC/SMC coculture is therefore considered to be the consequence of the elevated level of CNP. The natriuretic peptides are recognized to manifest their biological actions through two subtypes of biologically active natriuretic peptide receptors, the ANP-A and ANP-B receptors.4 The ANP-A receptor is activated by ANP and BNP, and the ANP-B receptor is selectively activated by CNP.5 6 We demonstrated that SMCs predominantly express the ANP-B receptor, whereas ECs express almost exclusively the ANP-A receptor.28 The elevated level of cGMP production in the EC/SMC coculture was comparable to that seen in SMCs exposed to 10 nmol/L CNP, while 10 nmol/L CNP gave no effect on cGMP production in ECs.28 Taken together, these results clearly indicate that endothelial CNP actually acts on the ANP-B receptor of SMCs to induce cGMP elevation in the EC/SMC coculture. Previously, we demonstrated the coexpression of genes for CNP and the ANP-B receptor in human blood vessels in vivo and suggested the existence of the vascular natriuretic peptide system.7 The present results can serve as in vitro evidence that CNP can act locally as a vascular natriuretic peptide within blood vessels.
The increase of the intracellular cGMP level in the EC/SMC coculture obtained in the present study was similar to that found in previous reports.16 29 Several lines of evidence suggest that NO and a labile NO-containing substance are generated by vascular ECs from the amino acid L-arginine and that they modulate the tone of the underlying SMCs by stimulating the soluble form of guanylate cyclase, resulting in elevation of the cGMP concentration.30 The authors of the previous works claimed that the increase of the cGMP level in their EC/SMC coculture system was therefore due to the generation of NO. Our study demonstrates, however, that elevated cGMP accumulation is mainly dependent on endothelial CNP in our coculture model, since anti-CNP monoclonal antibody and HS-142-1 dramatically reduced cGMP accumulation. Our KY-CNP-I is demonstrated to be specific to CNP, and HS-142-1 was reported to have no effect on cGMP formation by NO.21 The apparent discrepancy between our study and previous reports can be partly explained by the fact that our study examined relatively long effects of the coculture on cGMP production. NO is such a short-lived humoral factor that its effect may be dismissed in our system. To evaluate the contribution of NO, further experiments using NO-generation inhibitors such as nitro-arginine are needed.
We could detect an appreciable level of active TGF-ß in the EC/SMC coculture with direct contact, which confirms the previous reports that TGF-ß is released and/or activated by the EC/SMC coculture.26 31 The present study also showed that the activation of TGF-ß actually occurs in the direct interaction of ECs and SMCs, while the total production of TGF-ß is apparently not altered between SMCs alone and the EC/SMC coculture. Although the previous reports showed that ECs produce a latent form of TGF-ß,26 31 the level of TGF-ß in our EC culture was below the detection limit. This may in part reflect the difference of culture conditions and cell species.
The level of TGF-ß in the EC/SMC coculture is, as we demonstrated previously, sufficient to stimulate endothelial production of CNP.11 In addition, augmentation of endothelial CNP secretion was observed only in the EC/SMC coculture with direct contact, in which TGF-ß activation occurred. In the present study, TGF-ß (100 pmol/L) augmented the expression of CNP mRNA of ECs alone but produced no effect on that of SMCs alone. Furthermore, we also showed that increased intracellular cGMP accumulation in the EC/SMC coculture was abrogated by the neutralizing antibody against TGF-ß. These results together indicate that the effect of EC/SMC coculture on CNP production appears to be at least in part the result of the formation of the active TGF-ß and that locally activated TGF-ß stimulates endothelial CNP gene expression in the direct contact between ECs and SMCs.
The present study demonstrated that [3H]thymidine incorporation in cells treated with the conditioned medium from ECs stimulated by TGF-ß was significantly increased by treatment with KY-CNP-I and HS-142-1. The conditioned medium from ECs itself had no significant effect on SMC growth in the present study. The conditioned medium collected from ECs should contain a number of growth stimulators and growth inhibitors besides CNP. It is considered that the summation of the growth-regulatory activity of these factors in the conditioned medium showed no apparent effect on SMC growth.
Furthermore, in the present study, blockade of endogenous CNP action by KY-CNP-I and HS-142-1 significantly enhanced the cell number in the EC/SMC coculture. Since ECs express almost exclusively the ANP-A receptor and SMCs express predominantly the ANP-B receptor, the increase of the cell number in the coculture can be interpreted to be due to enhanced proliferation of SMCs. Taken together, the results of the present study indicate that endothelial CNP at the increased level actually inhibits the proliferation of SMCs through the elevation of intracellular cGMP concentration.32 33
The involvement of the clearance receptor in the antimitogenic action of natriuretic peptides is a recent controversial issue. Previously, we used C-ANF(4-23), which is the specific agonist for the clearance receptor, and demonstrated the lack of growth-inhibitory property of this peptide.12 We and others also reported that 8-bromo-cGMP exerted antiproliferative action.32 33 In contrast, Cahill and Hassid34 35 used three kinds of C-receptor agonists, namely C-ANF(4-23), [Cys105, Cys121]rANF(104-126), and Cys116-rANF(102-116)-NH2. They demonstrated that the latter two C-receptor agonists inhibited SMC growth as potently as ANP [rANF(99-126)]. They also observed that C-ANF(4-23) does not exert antiproliferative action. In addition, they showed that C-ANF(4-23) antagonized the antiproliferative action of ANP and their two C-receptor agonists. Thus, the involvement of the clearance receptor or cGMP-independent pathway for vascular growth suppression by natriuretic peptides still requires further clarification. So far, we think that cGMP-mediated growth inhibition of SMCs is (at least in part) responsible for CNP-induced vascular growth inhibition.
It has been suggested that the phenotypic change of SMCs in vivo is one
of the key developments related to the pathophysiology of certain
vascular diseases, including atherosclerosis,
hypertension, and restenosis following vessel angioplasty
and bypass grafting.36 We previously demonstrated that the
contractile phenotype of SMCs, which is generally thought to be
associated with cell contractility, expresses mainly
the ANP-A receptor, while the synthetic phenotype of SMCs,
which has been supposed to be implicated in proliferative vascular
changes, expresses the ANP-B receptor more abundantly than the ANP-A
receptor.8 It has also been recently reported that the
ANP-B receptor expression is upregulated in rat carotid arteries after
vascular injury.37 Furthermore, Fujio et al38
also reported possible gene regulation of the ANP-A and ANP-B receptors
by TGF-ß. Since SMCs in our coculture system possess a synthetic
phenotype to express the ANP-B receptor predominantly, this
EC/SMC coculture system seems likely to facilitate the study of the
interaction of ECs and SMCs as an in vitro model of proliferative
vascular change. The results of the present study using the
coculture system together with our previous observations suggest that
the vascular natriuretic peptide system is
activated in the proliferative vascular lesion so that
endothelial CNP at an enhanced level can inhibit
further proliferation of SMCs through the upregulated ANP-B receptor.
The vascular natriuretic peptide system could thus play a
protective role in the development of the proliferative vascular lesion
(Fig 8).
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The importance of reendothelialization of the intimal hyperplasia after endarterectomy or anastomotic thickening has been pointed out39 ; however, the actual in vivo relevance is still controversial.40 Therefore, the significance of CNP in in vivo proliferative vascular lesions requires further investigation.
In conclusion, the present study demonstrates that endothelial production of CNP is augmented by the coculture of ECs with SMCs and that cGMP generation of SMCs in the coculture is increased by CNP released from ECs. Endothelial production of CNP and the subsequent increase of cGMP production in the EC/SMC coculture system is at least in part regulated by activation of TGF-ß. Endothelial CNP was shown to have an inhibitory effect on the proliferation of SMCs. This is evidence for the possible significance of the vascular natriuretic peptide system, implicating CNP and the ANP-B receptor in the interaction of ECs and SMCs for the regulation of vascular growth.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported in part by research grants from the Japanese Ministry of Education, Science, and Culture; the Japanese Ministry of Health and Welfare Disorders of Adrenal Hormone research committee; the Molecular Approach for the Pathogenesis of Immunological Disorder research committee; the Smoking Research Foundation; the Yamanouchi Foundation for Research on Metabolic Disorders; the Salt Science Research Foundation; the Uehara Memorial Foundation; and the Japanese Society for Cardiovascular Diseases. We thank Hisayo Kitoh, Mihoko Shida, Katsuko Sasamoto, and Ayumi Takagoshi for their excellent secretarial work.
Received August 21, 1995; accepted December 22, 1995.
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