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Endocrinology Vol. 139, No. 4 1920-1926
Copyright © 1998 by The Endocrine Society

ARTICLES

Regulation of Endothelial Production of C-Type Natriuretic Peptide by Interaction between Endothelial Cells and Macrophages1

Shin-ichi Suga, Hiroshi Itoh, Yasato Komatsu, Hiroshi Ishida, Toshio Igaki, Jun Yamashita, Kentaro Doi, Tae-Hwa Chun, Takaaki Yoshimasa, Issei Tanaka and Kazuwa Nakao

Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, and the Department of Internal Medicine and Clinical Research Center (H.I.), National Utano Hospital, Kyoto, Japan

Address all correspondence and requests for reprints to: Hiroshi Itoh, M.D., Ph.D., Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin kawaharacho, Sakyo-ku, Kyoto 606, Japan. E-mail: hiito{at}kuhp.kyoto-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We demonstrated endothelial production of C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, and its regulation by cytokines, including tumor necrosis factor-{alpha} (TNF{alpha}). We thus proposed that CNP can control vascular tone and growth as an endothelium-derived relaxing peptide. We also revealed the marked elevation of plasma CNP concentration in patients with septic shock, in which TNF{alpha} plays a significant part. As the interaction between endothelial cells (EC) and monocytes-macrophages plays a pivotal role in the pathogenesis of atherosclerosis, we investigated the effect of coculture of EC and macrophages on endothelial production of CNP. We used a human monocytic leukemia cell line, THP-1, which differentiates into macrophages when treated with phorbol 12-myristate 13-acetate. The coculture of EC and THP-1-derived macrophages enhanced CNP secretion by more than 10-fold compared with the single culture of EC or the coculture of EC and THP-1 without phorbol 12-myristate 13-acetate treatment. Prevention of direct contact between EC and THP-1-derived macrophages did not attenuate the increase in CNP secretion. Northern blotting revealed the augmentation of CNP messenger RNA expression in EC in the coculture. We detected TNF{alpha} in the conditioned medium from the coculture of EC and THP-1-derived macrophages. Furthermore, anti-TNF{alpha} antibody inhibited the stimulation of CNP secretion in the coculture. CNP at a concentration of 1 nM did not stimulate cGMP production in EC or THP-1-derived macrophages, but it elevated cGMP production significantly in vascular smooth muscle cells. These results indicate that endothelial production of CNP is stimulated mainly by TNF{alpha} released from THP-1-derived macrophages in the coculture. Endothelial CNP at the enhanced level may be one of the vascular mediators to regulate local vascular tone and growth through cGMP production by vascular smooth muscle cells, suggesting the potential significance of endothelial CNP in atherosclerosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE NATRIURETIC peptide family comprises at least three endogenous peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), and is considered to be responsible for the regulation of cardiovascular and body fluid homeostasis (1, 2). ANP and BNP act mainly as cardiac hormones, secreted from the atrium and ventricle of the heart, respectively. CNP, the third member of the natriuretic peptide family, was originally isolated from the porcine brain (3). CNP with 22 amino acid residues has a ring structure formed by an intramolecular disulfide bond, which is conserved in ANP and BNP. An N-terminally elongated form of CNP with 53 amino acid residues (CNP-53) was also identified in the porcine brain (4). In contrast to ANP and BNP, CNP was claimed to act principally as a neuropeptide (3, 5). However, we demonstrated endothelial production of CNP (6) and the in vivo gene expression of CNP and its specific receptor, the ANP-B receptor, in the intact blood vessels (7). CNP was shown to have a potent growth inhibitory action on vascular smooth muscle cells (VSMC) (8, 9). In addition, endothelial production of CNP is regulated by several growth factors and cytokines (6, 10). Among them, transforming growth factor-ß and tumor necrosis factor-{alpha} (TNF{alpha}) are potent stimulators of CNP production. It is also demonstrated that CNP is present in human plasma and that the plasma CNP concentration is elevated in patients with septic shock. (11). Thus, we proposed the existence of the vascular natriuretic peptide system, in which CNP can induce relaxation and growth inhibition of VSMC through the elevation of cGMP produced by particulate guanylate cyclase (the ANP-B receptor).

Accumulating evidence indicates the significance of complicated interaction among vascular cells, that is endothelial cells (EC), VSMC, and macrophages, in the regulation of vascular function and remodeling. Indeed, the interaction between EC and macrophages plays a pivotal role in atherogenesis. In the early lesions of atherosclerosis, endothelial cell dysfunction caused by several sources of injury stimulates the adhesion of monocytes to EC and their migration into the intima, where they differentiate into macrophages. When exposed to agonists such as oxidized low density lipoprotein (LDL) in the intima, macrophages produce various growth regulatory molecules and chemoattractants. The autocrine and paracrine actions of these molecules can alter vascular cell functions and eventually contribute to the progression of atherosclerotic lesions (12, 13, 14).

In the present study, to elucidate the physiological and pathophysiological significance of endothelial CNP in atherogenesis, we examined the effects of the interaction between EC and macrophages on endothelial production of CNP, using the coculture technique.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
EC were isolated from the bovine carotid artery as we previously reported (6). The cells were cultured in 100-mm culture dishes in DMEM containing 10% FCS. Cells were identified as EC by phase microscopic "cobblestone appearance" and their ability to take up acetylated LDL (6). Experiments were performed using EC between passages 12–20. A human monocytic leukemia cell line, THP-1, was obtained from the Japan Cell Research Bank (Tokyo, Japan) and was passaged at concentrations of 2–3 x 105 cells in 75-cm2 culture flasks in RPMI 1640 supplemented with 10% FCS (15). VSMC were obtained from the thoracic aorta of male Wistar rats and cultured in DMEM with 10% FCS as previously reported (16). All cultures were allowed to grow at 37 C in a humidified atmosphere containing 5% CO2.

Coculture
THP-1 is known to differentiate into macrophages after treatment with phorbol ester (15, 17). Therefore, we established the coculture of EC and macrophages (EC/macrophage coculture) using THP-1 treated with phorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co., St. Louis, MO). To induce differentiation into macrophages, THP-1 were incubated with 100 nM PMA as previously reported (15, 18). EC were cocultured with THP-1 using two methods, one in which direct contact between EC and THP-1 was allowed and another in which direct contact was prevented.

Coculture with direct contact. EC were grown to confluence in 24-well culture plates or 100-mm culture dishes (Costar Corp., Cambridge, MA). The confluent monolayers were washed with serum-free DMEM, and various numbers of THP-1 harvested in DMEM containing 0.5% FCS were seeded on the EC monolayer. Then, the cells were incubated with or without PMA for the indicated time at 37 C. Single cultures of EC and THP-1 were performed in the presence or absence of PMA, which served as the control.

Coculture without direct contact. Additional coculture study was performed to investigate the role of contact between EC and THP-1. In this experiment, EC and THP-1 were cultured in the same well, but the direct contact between these two cell types was prevented by plating THP-1 on micropore filters (Transwell, Costar Corp.), which were inserted into wells of 24-well plates or the 100-mm culture dishes and sited 1–2 mm above EC. The micropore filter was formed by cellulose membrane containing 0.4-µm pores, which allow diffusion of soluble substances. All other experimental conditions were identical to those described in the coculture with the direct contact.

In some experiments, confluent endothelial cells in 24-well plates were incubated with recombinant human TNF{alpha} (donated by Dainippon Pharmaceutical Co., Osaka, Japan). Conditioned media from each experiment were collected and centrifuged at 600 x g for 10 min. The supernatants were stored at -20 C until the RIA or enzyme-linked immunosorbent assay (ELISA).

RIA for CNP
The RIA for CNP was performed as we previously reported (5, 6). The cross-reactivities with {alpha}ANP, human BNP, bovine BNP, and CNP-53 were 0.2%, less than 0.01%, 14%, and 100% on a molar basis, respectively.

RIA for endothelin (ET)
Measurement of ET-1 concentrations was performed as previously described (19). The cross-reactivities with ET-2, ET-3, and human big ET-1 in the RIA were 80%, 20%, and 80% on a molar basis, respectively.

ELISA for TNF{alpha}
The concentrations of human TNF{alpha} in the conditioned media were determined with a sandwich ELISA method using monoclonal antibodies to TNF{alpha}, as previously reported (20, 21). The detection limit for TNF{alpha} of this assay was 10 pg/ml.

High performance-gel permeation chromatography (HP-GPC)
HP-GPC was performed on a TSK-GEL G2,000 SW column (7.5 x 600 mm; Toyo Soda, Tokyo, Japan) eluted with 10 mM trifluoroacetic acid containing 0.3 M sodium chloride and 30% acetonitrile as a solvent, as we previously reported (6).

RNA extraction and Northern blot analysis
Cultured cells in 100-mm culture dishes or on micropore filters for a 100-mm dish were washed twice with Dulbecco’s PBS. RNA were extracted by the guanidinium thiocyanate CsCl method and were subjected to polyadenylated [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 by cDNA synthesis and the PCR method as we previously reported (6). The human G3PDH probe was purchased from Clontech (no. 9205-1, Palo Alto, CA) (22). The filters were hybridized with the 32P-labeled probe at 42 C in 50% formamide, 5 x SSC, 5 x Denhardt’s reagent, 50 mM sodium phosphate buffer (pH 6.8), 0.1% SDS, and 100 µg/ml heat-denatured salmon sperm DNA and washed at 65 C in 0.1 x SSC and 0.1% SDS (6, 9). Autoradiography was performed on x-ray films with intensifying screens.

Neutralization of CNP secretion
Rabbit antihuman TNF{alpha} serum was provided by Dainippon Pharmaceutical Co. (23). The appropriate antibody dilution was determined by inhibiting the activity of 40 ng/ml TNF{alpha}. The antibody diluted by 1:1000 completely neutralized the cytotoxic activity of TNF{alpha} on L-M cells, measured by the dye uptake method (24). Nonimmune rabbit serum was purchased from Zymed Laboratories (San Francisco, CA). Either anti-TNF{alpha} antiserum or nonimmune serum was added to the culture medium at the beginning of the coculture. After 24-h incubation, CNP concentrations in the conditioned medium were measured.

Determination of intracellular cGMP in cultured cells
EC, THP-1, and VSMC were cultured in 24-well plates and washed with serum-free DMEM. The cells were preincubated for 10 min with 225 µl DMEM containing 0.1% BSA and 0.5 mM isobutylmethylxanthine. Then, various concentrations of CNP or ANP were added to the medium, and the cells were incubated for 5 min at 37 C. The medium was then rapidly aspirated, and 1 ml ice-cold 6% trichloroacetic acid was added to each well. Intracellular cGMP concentrations were measured using specific RIA for cGMP, as we previously described (25).

Peptides
Human ANP and CNP were purchased from The Peptide Institute (Minoh, Japan).

Statistics
Values are expressed as the mean ± SEM. Statistical analyses were performed using Student’s t test or one-way ANOVA. A significant difference was defined as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of EC/macrophage coculture on CNP secretion
Coculture with direct contact. We examined the effect of the coculture of EC and macrophages on CNP secretion. Figure 1Go depicts the time course of CNP-like immunoreactivity (CNP-LI) concentrations in the conditioned medium. In the single culture of EC, the CNP-LI concentration increased time dependently and reached a plateau at 24 h (24.0 ± 2.2 fmol/well). The addition of 100 nM PMA caused a significant increase in the accumulation of CNP-LI by 2-fold (46.0 ± 2.2 fmol/well) at 24 h in the single culture of EC, consistent with our previous observation (6). Incubation with 100 nM PMA induced differentiation of THP-1 into macrophages as previously reported (15, 18). However, CNP-LI was undetectable (<10 fmol/well) up to 48 h in the conditioned medium from the single culture of THP-1 with or without PMA treatment. The coculture of EC and THP-1 had no significant effect on the CNP-LI concentrations in the conditioned medium until 48 h compared with those from the single culture of EC without PMA treatment. In marked contrast, the coculture of EC and THP-1 with PMA treatment, that is the coculture of EC and THP-1-derived macrophages, augmented the accumulation of CNP-LI within 6 h after establishment of the coculture. The CNP-LI concentration, which reached a plateau at 24 h, was 20-fold higher (435 ± 24 fmol/well·24 h) than that in the single culture of EC or in the coculture of EC and THP-1. The coculture of EC and THP-1 significantly decreased the ET-LI concentration in the conditioned medium (3.84 ± 0.86 pmol/well·24 h in EC/THP-1 coculture vs. 7.63 ± 0.25 pmol/well·24 h in the single culture of EC; n = 4). In contrast to CNP secretion, ET secretion was not different between EC/THP-1 coculture and EC/THP-1-derived macrophage coculture (4.05 ± 0.56 pmol/well·24 h; n = 4).



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  		Figure 1. Time course of CNP-LI concentrations in the conditioned medium of the single culture of EC or the coculture. In the single culture, 2 x 105/well EC were incubated in the absence ({circ}) or presence ({blacktriangleup}) of 100 nM PMA. In the coculture, 2 x 105/well EC and 5 x 105/well THP-1 were cultured with (•) or without ({triangleup}) 100 nM PMA. Each point represents the mean ± SEM of three separate experiments (n = 4–5). *, P < 0.05 vs. single culture of EC without PMA.

 
To evaluate the influence of the THP-1/EC ratio on CNP secretion from the coculture, we cocultured EC with THP-1 or THP-1-derived macrophages for 24 h at THP-1/EC ratios of 0.625:1, 1.25:1, and 2.5:1. Although the number of THP-1 did not affect CNP secretion in the coculture (Fig. 2AGo), the elevation of CNP secretion was dependent on the number of THP-1-derived macrophages (Fig. 2BGo). The degree of stimulation of CNP secretion increased with increasing number of THP-1-derived macrophages, and significant stimulation was observed at 1.25 x 105 cells/well.



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  		Figure 2. Effect of the number of THP-1 (A) or THP-1-derived macrophages (B) on CNP-LI secretion from the coculture. EC (2 x 105 cells/well) were incubated with different number of THP-1 or THP-1-derived macrophages for 24 h. Each point represents the mean ± SEM of three or four separate experiments (n = 4). *, P < 0.05 vs. single culture of EC.

 
Figure 3Go shows the HP-GPC profiles of CNP-LI in the conditioned medium of the single cultures of EC and EC/THP-1-derived macrophage coculture. The CNP-LI in the conditioned medium of EC (Fig. 3AGo) and the CNP-LI in the conditioned medium of the coculture (Fig. 3BGo) were composed of two components corresponding to CNP and CNP-53, as we previously reported (6).



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  		Figure 3. A typical HP-GPC profile of CNP-LI in the conditioned medium from the single culture of EC (A) and EC/THP-1-derived macrophage coculture (B). Arrows denote the elution positions of synthetic CNP-53 and CNP.

 
Coculture without direct contact. To determine the requirement for contact between EC and THP-1-derived macrophages for the augmentation of CNP secretion from the coculture, direct contact or cell proximity between EC and THP-1-derived macrophages was prevented by a micropore filter, which allowed the exchange of diffusible substances. Figure 4Go reveals that EC/THP-1-derived macrophage coculture without direct contact also stimulated the accumulation of CNP-LI (450 ± 34 fmol/well·24 h) in the conditioned medium, similar in magnitude to that in the coculture with direct contact.



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  		Figure 4. CNP-LI accumulation in the conditioned medium 24 h after the initiation of the coculture with or without direct contact. Column 1, Single culture of EC; column 2, single culture of EC with 100 nM PMA; column 3, coculture of EC and THP-1 with direct contact; column 4, coculture of EC and THP-1-derived macrophages with direct contact; column 5, coculture of EC and THP-1 without direct contact; column 6, coculture of EC and THP-1-derived macrophages without direct contact. In the coculture without direct contact, THP-1 or THP-1-derived macrophages were cultured on micropore filters. The numbers of EC and THP-1 or THP-1-derived macrophages were 2 x 105 and 5 x 105/well, respectively. Each point represents the mean ± SEM of four separate experiments (n = 4). *, P < 0.05, vs. single culture of EC.

 
Expression of CNP messenger RNA (mRNA) in coculture
Northern blot analysis detected CNP mRNA with a size of 1.2 kilobases in a limited quantity in single culture of EC with or without PMA treatment (Fig. 5Go, lanes 1 and 2) as we previously reported (6, 9). CNP mRNA was undetectable in poly(A)+ RNA extracted from THP-1 (lane 3) or THP-1-derived macrophages (lane 4). We were unable to detect CNP mRNA in EC/THP-1 coculture (lane 5); however, the expression of CNP mRNA was augmented in EC/THP-1-derived macrophage coculture with direct contact (lane 6). To determine which cell populations produce CNP in the coculture, we measured the CNP mRNA levels of EC and THP-1-derived macrophages in the coculture without direct contact. Although CNP mRNA was not detected in THP-1-derived macrophages from the coculture (lane 8), the expression of CNP mRNA increased markedly in EC from the coculture (lane 7). Glyceraldehyde-3-phosphate dehydrogenase mRNA expression was not different among different RNA samples.



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  		Figure 5. Northern blot analysis of CNP mRNA in the coculture. RNA was isolated 9 h after the initiation of the coculture and poly(A)+ RNA (5 µg) was analyzed using the CNP cDNA as a probe. Lane 1, Single culture of EC; lane 2, single culture of EC with 100 nM PMA; lane 3, single culture of THP-1; lane 4, single culture of THP-1-derived macrophages; lane 5, EC/THP-1 coculture with direct contact; lane 6, EC/THP-1-derived macrophage coculture with direct contact; lane 7, EC from EC/THP-1-derived macrophage coculture without direct contact; lane 8, THP-1-derived macrophages from EC/THP-1-derived macrophage coculture without direct contact. THP-1-derived macrophages were cultured on micropore filters in the coculture without direct contact.

 
TNF{alpha} concentrations in conditioned medium of coculture
As TNF{alpha} is one of the most potent stimulators of endothelial secretion of CNP (10), we investigated the possibility that TNF{alpha} was the mediator to augment endothelial production of CNP in the coculture. TNF{alpha} was not detected (<10 pg/ml) in the conditioned medium from the single culture of EC with or without PMA treatment. TNF{alpha} was also undetectable in the conditioned medium from EC/THP-1 coculture. In contrast, we could detect TNF{alpha} in the conditioned medium from EC/THP-1-derived macrophage coculture (66 ± 8 pg/ml; n = 4) 12 h after the establishment of the coculture. In addition, we confirmed that when 100 pg/ml TNF{alpha} was added exogenously to the single culture of EC, it induced a 2-fold increase in CNP secretion.

Neutralization of stimulated CNP secretion
We examined the effect of a neutralizing anti-TNF{alpha} serum on endothelial secretion of CNP. Figure 6Go shows the result obtained when EC/THP-1-derived macrophage coculture was incubated in the presence of anti-TNF{alpha} serum or control nonimmune serum. Neither control serum nor anti-TNF{alpha} serum affected CNP secretion from the single culture of EC. Although control serum had no inhibitory effect on elevated CNP secretion in the coculture, inclusion of anti-TNF{alpha} serum blocked the elevation of CNP secretion by more than 90% at a dilution of 1:1000. In contrast, anti-TNF{alpha} serum did not affect the decrease in ET secretion induced by EC/THP-1 coculture or EC/THP-1-derived macrophage coculture.



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  		Figure 6. Effect of anti-TNF{alpha} antibody on CNP secretion from EC/THP-1-derived macrophage coculture. EC (2 x 105/well) were cultured with THP-1-derived macrophages (5 x 105/well) for 24 h in the presence of control nonimmune rabbit serum (1:1000 dilution) or anti-TNF{alpha} antiserum (1:1000 dilution). Each point represents the mean ± SEM of three separate experiments (n = 4). *, P < 0.05, vs. corresponding control serum.

 
cGMP production by CNP and ANP in cultured vascular cells
To investigate the possible target cells of CNP secreted from EC, intracellular cGMP accumulation by CNP and ANP were examined in EC, THP-1-derived macrophages, and VSMC. ANP (100 nM) significantly stimulated cGMP production in EC (Fig. 7AGo) and THP-1-derived macrophages (Fig. 7BGo); however, CNP at concentrations up to 100 nM had no stimulatory effect on cGMP production in these cell types. The patterns of cGMP production by CNP and ANP are consistent with those via the activation of the ANP-A receptor, as we reported previously (25, 26). By contrast, in VSMC (Fig. 7CGo), 1 nM CNP potentiated cGMP production by 3-fold, and CNP was a more potent stimulator of cGMP production than was ANP. The patterns of cGMP production by CNP and ANP in VSMC are compatible with those mediated via the ANP-B receptor (25, 26).



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  		Figure 7. cGMP production by CNP and ANP in cultured EC (A), THP-1-derived macrophages (B), and VSMC (C). Each point represents the mean ± SEM of three separate experiments (n = 4). *, P < 0.05 vs. vehicle.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using the coculture of EC and THP-1-derived macrophages, the present study demonstrates that macrophages markedly augment the endothelial production of CNP. In the absence of PMA-induced differentiation of THP-1 into macrophages, EC/THP-1 coculture had no significant effect on CNP production. PMA treatment induced a mild increase in CNP secretion in the single culture of EC. However, the potency to stimulate CNP production was far weaker than that in EC/THP-1-derived macrophage coculture. The elevation of CNP secretion in the coculture depended on the number of THP-1-derived macrophages. In addition, the differentiation of THP-1 into macrophages by PMA treatment did not affect ET secretion in the coculture. Taken together, our results indicate that the stimulation of CNP production in EC/THP-1-derived macrophages coculture is a specific event.

The regulation of CNP secretion by THP-1-derived macrophages was independent of direct cell-cell contact or cell proximity; that is, the coculture without direct contact showed an increase in CNP secretion comparable to that in the coculture with direct contact. This finding suggests the existence of diffusible factors to mediate the stimulation. When THP-1 cells differentiate into macrophages by PMA, they undergo an alteration in cell morphology, acquire the capacity to produce several secretory products such as cytokines and enzymes, and change the expression of protooncogenes, membrane antigens, and receptors, (17, 18, 27). Among these factors, we focused on cytokines, especially TNF{alpha}, because we demonstrated that it is one of the most potent stimulators of endothelial production of CNP (10). We could detect a considerable amount of TNF{alpha} in the conditioned medium of EC/THP-1-derived macrophage coculture. In addition, incubation with a similar amount of recombinant human TNF{alpha} enhanced endothelial production of CNP. Furthermore, neutralizing antihuman TNF{alpha} serum abrogated the elevation of CNP production in the coculture. These results indicate that TNF{alpha} is the principal mediator of stimulation of CNP production in coculture. It is also important to elucidate the cellular origin of CNP in the coculture, because there is a report that THP-1 synthesizes CNP (28). The elevation of CNP production in the coculture without direct contact enabled us to evaluate CNP production by each cell type separately. Although CNP mRNA was undetectable in both THP-1 and THP-1-derived macrophages, the CNP mRNA level increased tremendously in EC in the coculture without direct contact. Thus, the present study clearly demonstrates that EC/THP-1-derived macrophage coculture stimulates endothelial production of CNP by the release of TNF{alpha} from THP-1-derived macrophages.

The reason why ET secretion was suppressed by the coculture of EC and THP-1 is not known at present. There was no difference in ET secretion between that in EC/THP-1 coculture and that in EC/THP-1-derived macrophage coculture. Therefore, it can be at least considered that the increase in CNP secretion in EC/THP-1-derived macrophage coculture was not a nonspecific event. As the decrease in ET secretion in EC/THP-1 coculture should also be implicated in vascular remodeling, the elucidation of its mechanism deserves further investigation.

The natriuretic peptides are recognized to exert their biological actions through the elevation of intracellular cGMP by activating the biologically active receptors, the ANP-A and ANP-B receptors. ANP and BNP mainly stimulate the ANP-A receptor, whereas CNP is a selective ligand for the ANP-B receptor (25, 26). In the present study, the CNP concentration was approximately 1 nM in the conditioned medium of EC/THP-1-derived macrophage coculture. Although CNP at this concentration failed to potentiate cGMP production in THP-1-derived macrophages and EC, it activated cGMP production in cultured VSMC. In addition, Drewett et al. reported that 1 nM CNP can induce relaxation of precontracted aorta (29). Combined with our previous report on the coexpression of CNP and the ANP-B receptor in vascular wall in vivo (7), the present study can serve as evidence to show that CNP could be a novel mediator of macrophage/EC/VSMC interaction and further supports the existence of the vascular natriuretic peptide system (6).

Recently, Vollmer et al. reported that murine peritoneal and bone marrow macrophages produce CNP and that CNP production by macrophages is stimulated by immunomodulators, such as lipopolysaccharide and zymosan (30). In humans, there is a recent report on the existence of CNP not only in EC, but also in macrophages of atherosclerotic lesions (31). We could not detect CNP production by THP-1-derived macrophages in the present study. In human coronary arteries, however, not all macrophages were CNP positive (31), which suggests that various factors may participate in the regulation of CNP production in macrophages, as in the case of endothelial production of CNP (6, 9, 10). Therefore, it is possible that the THP-1-derived macrophages we used could secrete CNP under the existence of different stimuli. The other possibility is the difference in the ability of secretory functions between THP-1-derived macrophages and native macrophages. It has been reported that THP-1-derived macrophages behave like native monocyte-derived macrophages, including the production of cytokines and peptide hormones, which allows us to use them widely to study varying aspects of macrophage biology (17, 32). Nevertheless, there may be some discrepancy in CNP secretion between THP-1-derived macrophages and native macrophages. Further interest should focus on the regulation of CNP production by macrophages.

Monocyte-derived macrophages are present and play significant roles throughout all stages of atherogenesis (12, 13, 14). Endothelial dysfunction can induce the migration of monocytes into the intima, where they differentiate into macrophages. Upon exposure to agonists, such as oxidized LDL, macrophages are activated to release growth regulatory molecules, including TNF{alpha}, interleukin-1, and transforming growth factor-ß, which, by inducing the expression of platelet-derived growth factor in EC and VSMC, could be growth stimulators for VSMC. As a potential balance to these atherogenic factors, they secrete several molecules that attenuate the growth of VSMC and/or inhibit adhesion of mononuclear cells to EC. Cell culture studies have revealed that CNP inhibits the growth of VSMC (8, 9). We previously demonstrated that the expression of the ANP-B receptor, the selective receptor for CNP, increases in VSMC in the synthetic phenotype, which play a critical role in atherogenesis (16), and suggested the protective role of CNP against atherosclerosis (9). Indeed, administration of CNP could suppress the intimal thickening of the injured carotid arteries (33). Thus, the present study presents in vitro evidence that endothelial CNP could be a new antiatherogenic factor that potentially antagonizes the TNF{alpha}-mediated growth-promoting effect of macrophages on VSMC.

Patients with hyperlipidemia or hypertension have impaired endothelial functions, including the deficiency of endothelial nitric oxide production (34, 35). It has been thought that the impairment of vasorelaxant, antiproliferative, and antithrombotic actions of nitric oxide may initiate or promote atherosclerosis in these patients (36). Interestingly, the proportion of CNP-positive EC decreases in association with the progression of atherosclerotic lesions in human (31). Moreover, we reported that oxidized LDL and low shear stress suppress endothelial secretion of CNP (37, 38). Therefore, it is possible to hypothesize that endothelial injury might also impair endothelial production of CNP in basal state or in response to mitogenic stimuli for VSMC, which could be one of the causes for unregulated proliferation of VSMC.

In conclusion, the present investigation demonstrates that endothelial production of CNP is potently stimulated by macrophages principally through the release of TNF{alpha}. According to the results, CNP can be added to the list of compounds mediating macrophage/EC/VSMC interaction in vascular wall, which supports the idea that the vascular natriuretic peptide system could play some role in the pathogenesis of atherosclerosis.


   Acknowledgments
 
We thank Dainippon Pharmaceutical Co. (Osaka, Japan) for kindly providing TNF{alpha} and anti-TNF{alpha} serum, and Japan Cell Research Bank (Tokyo, Japan) for donating THP-1. We also thank Ms. Hisayo Kitoh for her secretarial assistance.


   Footnotes
 
1 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; Uehara Memorial Foundation; the Salt Science Research Foundation; Smoking Research Foundation; Yamanouchi Foundation for Research on Metabolic Disorders; and Tanabe Medical Frontier Conference. Back

Received August 18, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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