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
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-β (TGF-β) 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-β 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-β. Furthermore, the conditioned medium from ECs stimulated by TGF-β 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.
- cyclic GMP
- natriuretic peptide receptor
- endothelium-derived relaxing factor
- transforming growth factor-β
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-β, 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-α, augment CNP production as much as TGF-β.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.
Materials and Methods
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.
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 2×105 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 α-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, 5× SSC, 5× 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 0.1× 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-β and Neutralization of TGF-β Action
The level of TGF-β in the conditioned medium was determined by bioassay, using CCL-64 mink lung epithelial cells as described.23 The concentrations of TGF-β 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-β (human TGF-β, 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-β neutralizing antibody (described below).24 The concentrations of total TGF-β were estimated by acid activation of the conditioned medium as previously described.25
Rabbit anti-porcine TGF-β IgG was purchased from R&D Systems.26 This antibody has been shown to neutralize both porcine and human TGF-β. The dosage that produces 50% neutralization was determined to be 3 to 5 mg/L antibody in neutralization of 10 pmol/L TGF-β. Twenty milligrams per liter of either anti–TGF-β 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-β. 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 (1×105 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 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.
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.
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 (2×105 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.
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 (2×105 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.
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-β (100 pmol/L) for 12 hours (lane 2), while a weak band was detectable in either SMCs alone or SMCs exposed to TGF-β (100 pmol/L) (lanes 3 and 4, respectively). G3PDH mRNA expressions were not different in these lanes.
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).
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-β Activity in EC/SMC Coculture and Neutralization of TGF-β Action
TGF-β 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-β 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-β 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-β in the conditioned medium from the EC/SMC coculture (the numbers of ECs and SMCs were 2×105 cells per well) was five times higher than that from SMCs alone. Total TGF-β concentrations were similar between SMCs alone and the EC/SMC coculture (Table⇓).
Coincubation with 20 mg/L of the neutralizing antibody against TGF-β 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-β antibody elicited no significant effect on the cGMP levels in ECs alone or SMCs alone.
Furthermore, we compared active TGF-β concentration in the EC/SMC coculture with versus without direct contact, as described above (Fig 3⇑). Active TGF-β 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-β 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.
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±0.3×105 per well) compared with the control IgG–treated group (4.7±0.3×105 per well, P<.05). The administration of HS-142-1 (10 mg/L) also significantly increased the cell number in the coculture (6.5±0.3×105 per well) compared with the vehicle-treated group (5.4±0.2×105 per well, P<.05).
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-β in the EC/SMC coculture with direct contact, which confirms the previous reports that TGF-β is released and/or activated by the EC/SMC coculture.26 31 The present study also showed that the activation of TGF-β actually occurs in the direct interaction of ECs and SMCs, while the total production of TGF-β 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-β,26 31 the level of TGF-β 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-β 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-β activation occurred. In the present study, TGF-β (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-β. 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-β and that locally activated TGF-β 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-β 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-β. 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⇓).
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-β. 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.
Selected Abbreviations and Acronyms
|ANF||=||atrial natriuretic factor|
|ANP, BNP, and CNP||=||atrial, brain, and C-type natriuretic peptide, respectively|
|EC||=||vascular endothelial cell(s)|
|EC-CM||=||conditioned medium from ECs stimulated by TGF-β|
|SMC||=||vascular smooth muscle cell(s)|
|TGF||=||transforming growth factor|
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.
- © 1996 American Heart Association, Inc.
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