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(Hypertension. 1996;27:811-815.)
© 1996 American Heart Association, Inc.

Articles

Vascular Endothelial Growth Factor Suppresses C-Type Natriuretic Peptide Secretion

Kentaro Doi; Hiroshi Itoh; Yasato Komatsu; Toshio Igaki; Tae-Hwa Chun; Kazuhiko Takaya; Jun Yamashita; Mayumi Inoue; Takaaki Yoshimasa; Kazuwa Nakao

From the Department of Medicine and Clinical Science, Kyoto (Japan) University Graduate School of Medicine.

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. E-mail hiito@kuhp.kyoto-u.ac.jp.

	Abstract

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Abstract Angiogenesis plays a pivotal role not only in wound healing and tumor progression but also in diabetic angiopathy, arteriosclerosis, and collateral formation of obstructive vascular diseases. Vascular endothelial growth factor (VEGF) is now thought to be an endothelium-specific and potent angiogenic factor. We previously demonstrated that C-type natriuretic peptide (CNP), originally isolated from porcine brain, is produced by endothelial cells and proposed that CNP can exert control over vascular tone and growth as a local vascular regulator. In the present study, we examined the effect of VEGF on CNP secretion from endothelial cells using the specific radioimmunoassay for CNP we developed. VEGF (1 to 100 ng/mL) dose-dependently suppressed CNP secretion from cultured bovine endothelial cells, and 100 ng/mL VEGF suppressed endothelial CNP secretion to 28% of control levels (31.7±5.5 versus 8.9±0.8 fmol/mL, vehicle versus VEGF). VEGF also suppressed CNP mRNA expression in endothelial cells 9 hours after administration. In contrast, basic fibroblast growth factor (20 ng/mL), an endothelium-nonspecific angiogenic factor, significantly stimulated CNP secretion by 290%. These results indicate that VEGF can regulate vascular tone and growth in the process of angiogenesis through suppression of endothelial secretion of CNP, which is an endothelium-derived vasorelaxing and growth-inhibitory peptide.

Key Words: natriuretic peptides • endothelial growth factors • endothelial cell • cell division

	Introduction

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The natriuretic peptide family is composed of ANP, BNP, and CNP and is considered to be responsible for body fluid homeostasis and blood pressure regulation.^{1 2 3 4 5 6 7} We have demonstrated that ANP and BNP are mainly produced in the atrium and ventricle of the heart, respectively, and act as cardiac hormones.^{2 3 4} In contrast, CNP was originally discovered in the central nervous system and not detected in the heart^{8 9} and therefore was considered to act as a neuropeptide.^{7 10 11} However, we have recently demonstrated that CNP is produced in vascular ECs^{12 13} and that the endothelial secretion of CNP is regulated by various growth factors and cytokines, especially TNF- and TGF-ß. These results indicate the significance of CNP as an endothelium-derived relaxing peptide. We further demonstrated the gene expression of CNP and its specific receptor, the ANP-B receptor, in in vivo human blood vessels and reported the detection of CNP in human plasma.^{9 14 15} In addition, we and others have shown that natriuretic peptides can regulate the growth of vascular SMCs and ECs.^{16 17 18 19 20} Thus, we have proposed the existence of the vascular natriuretic peptide system, in which CNP could induce relaxation and growth inhibition of blood vessels through the elevation of cGMP produced by its specific receptor, the ANP-B receptor, which is the particulate guanylate cyclase itself.^{12 21 22}

In the vascular system, angiogenesis is an important process not only in the development and differentiation of embryonic vascular trees but also in somatic growth, wound healing, tissue and organ regeneration, and cyclic growth of the corpus luteum and endometrium.^{23 24 25 26} In addition, this process has important pathogenic effects in, for instance, tumor progression, arteriosclerosis, diabetic proliferative angiopathy, collateral formation of the occluded blood vessels, rheumatoid arthritis, psoriasis, and retrolental fibroplasia.^{24 25 26}

VEGF, a 34- to 42-kD heparin-binding dimeric disulfide–bonded glycoprotein, was isolated initially as a heparin-binding endothelial growth factor in the media conditioned by normal bovine pituitary folliculostellate cells and by a variety of transformed cell lines.^{27 28} VEGF was also purified independently as a tumor-secreted factor that induced vascular permeability 5000 times as potent as histamine; thus it was alternatively designated as vascular permeability factor (VPF). VEGF is unique among angiogenic growth factors by virtue of the fact that its two high-affinity receptors with tyrosine kinase domains (flt-1 and Flk-1/KDR) were present solely on ECs; consequently, VEGF can act as an EC-specific mitogen.^{29 30 31 32}

Previously, we and others reported that natriuretic peptides inhibit not only proliferation and hypertrophy of vascular SMCs but also proliferation of ECs through the augmentation of cGMP production in these cell types.^{16 17 18 19 20} Therefore, in the present study, we examined the action of VEGF on the endothelial secretion of CNP to elucidate the significance of the vascular natriuretic peptide system in the process of angiogenesis.

	Methods

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Cell Culture
ECs were isolated from adult bovine carotid artery by scraping the intimal surface with a scalpel and were cultured in DMEM (Nissei Pharmaceutical Co) supplemented with 10% FCS at 37°C in a humidified atmosphere containing 5% CO₂ as we have described.^{12 13} Cells at passages 15 through 25 were used. ECs were identified by the uptake of fluoresceinated acetylated low-density lipoprotein.

Growth Curves
For determination of cell numbers, ECs were grown to confluence in six-well culture dishes. The cells were replaced with 1.5 mL fresh DMEM containing 0.5% FCS and were incubated at 37°C with VEGF, bFGF, and vehicle. EC numbers were counted 48 hours after the addition of agents. Counts were performed by hemocytometer measurement immediately after cell harvest.

Determination of DNA Synthesis
Relative rates of DNA synthesis were assessed by determination of [³H]thymidine incorporation into trichloroacetic acid–precipitable material. Confluent ECs grown in 48-well culture dishes were pulsed for 6 hours with [³H]thymidine (2.5 mCi/mL), washed twice with cold phosphate-buffered saline and twice with 10% (wt/vol) cold trichloroacetic acid, and incubated with 10% trichloroacetic acid at 4°C for 30 minutes. Cells were then rinsed in ethanol (95%), dissolved in 0.25N NaOH at 4°C for 2 hours, and neutralized; radioactivity was determined by liquid scintillation spectrometry.¹⁶

Preparation of Conditioned Media
Conditioned media were prepared as we previously reported.^{12 13} ECs at 80% to 90% confluence were made quiescent by placement for 3 days in DMEM with 0.5% FCS. After 3 days of culture, ECs became just confluent. Confluent ECs in six-well plates were incubated with 1.5 mL DMEM containing 0.5% FCS at 37°C for the indicated time with or without the following agents: human VEGF (Pepro Tech Inc), human TNF- (Dainihon Pharmaceutical Co), porcine TGF-ß (R&D Systems, Inc), and human bFGF (R&D Systems, Inc). After incubation, conditioned media were collected and stored at -20°C until radioimmunoassay.

Radioimmunoassay for CNP
The radioimmunoassay specific for CNP was developed and performed as we previously reported.¹² Intra-assay and interassay coefficients of variation were 8.7% (n=9) and 9.1% (n=8), respectively.⁹ The cross-reactivities with -human ANP and human BNP were 0.2% and less than 0.01%, respectively, on a molar basis. For standardization of CNP secretion from ECs, we counted the EC number after sampling of conditioned media in each well. Then, we calculated endothelial CNP secretion per 10⁶ cells.

RNA Extraction
RNA was extracted from confluent bovine ECs by the guanidine thiocyanate/CsCl method^{12 14} and was subjected to poly(A)⁺ RNA enrichment.

Northern Blot Analysis for CNP mRNA
Northern blot analysis for CNP mRNA was performed as reported elsewhere,^{9 12 14} with the human CNP cDNA probe^{9 10} and human GAPDH probe (Clontech Laboratories, Inc).

Statistics
Values are expressed as mean±SE. Statistical analysis of data was performed by Student's t test or one-way ANOVA. Significant differences were defined at a value of P<.05.

	Results

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We first examined the mitogenic activity of VEGF on our ECs compared with that of bFGF. Cell numbers of ECs treated with vehicle, VEGF (10 and 30 ng/mL), and bFGF (20 ng/mL) were 46.6±3.3, 61.9±2.0, 84.8±1.4, and 62.08±1.5x10⁴ per well, respectively (n=4). It can be seen that VEGF and bFGF exerted comparable stimulatory potency for EC proliferation. [³H]Thymidine uptake of ECs in 0.5% FCS without any treatment was 46 126±1519 cpm per well, and [³H]thymidine uptake of ECs treated with VEGF (30 ng/mL) was 65 240±6132 cpm per well.

We examined the regulation of CNP secretion from ECs by VEGF. Fig 1 depicts the time course of CNP concentrations in the conditioned media of ECs treated with 10 ng/mL VEGF. CNP concentrations in EC-conditioned media increased in a time-dependent manner and reached a plateau at 24 hours in vehicle-treated ECs, as we reported.^{12 13} As clearly shown in Fig 1, 10 ng/mL VEGF significantly suppressed the increase of CNP concentration in EC-conditioned media. The suppressive action of VEGF on endothelial secretion of CNP was first observed 12 hours after the addition of VEGF. CNP concentration in EC-conditioned media was 31.7±5.4 fmol/10⁶ cells in the vehicle-treated group and 18.7±1.8 fmol/10⁶ cells in the VEGF-treated group (P<.05 versus vehicle) 48 hours after VEGF administration.

View larger version (12K): [in this window] [in a new window]   Figure 1. Time-course of CNP-like immunoreactivity (CNP-LI) concentrations in conditioned media of cultured ECs stimulated by VEGF. Cells were incubated with vehicle () or 10 ng/mL VEGF () (n=6). *P<.05 vs vehicle-treated cells.

Fig 2 depicts the dose-dependent effect of VEGF on CNP secretion from ECs. It can be seen that as little as 1 ng/mL VEGF induced a 15% decrease in CNP concentration in EC-conditioned media 48 hours after the addition of VEGF, and 10 ng/mL VEGF suppressed CNP secretion as much as 50% of the control value. No further decrease in CNP secretion was observed at larger VEGF doses. The ED₅₀ of the suppression of VEGF on CNP secretion was 2 ng/mL.

View larger version (16K): [in this window] [in a new window]   Figure 2. Dose-dependent effects of VEGF on CNP secretion from cultured bovine ECs. Cells were incubated with different VEGF concentrations for 48 hours (n=3). *P<.05 vs vehicle-treated cells. CNP-LI indicates CNP-like immunoreactivity.

Fig 3 and the Table show the effects of VEGF on TNF-– and TGF-ß–stimulated CNP secretion from ECs. As we reported previously, TNF- induced a 100-fold increase in CNP secretion 48 hours after incubation. VEGF at 30 ng/mL potently suppressed CNP secretion to less than 50% of the control level. A suppressive action of VEGF on TNF-–stimulated CNP secretion was also first observed 12 hours after incubation and persisted for the 48-hour observation period. Similarly, as we reported, TGF-ß (1 ng/mL) potently stimulated CNP secretion from ECs. VEGF (30 ng/mL) also suppressed augmented secretion of CNP from ECs treated with TGF-ß (1 ng/mL) by 55%.

View larger version (12K): [in this window] [in a new window]   Figure 3. Time course of effects of VEGF on CNP secretion augmented by TNF- (50 ng/mL). TNF- was added to the culture media of bovine ECs simultaneously with 30 ng/mL VEGF. CNP concentrations in media were determined 12, 24, and 48 hours after addition of agents (n=4). indicates TNF- (50 ng/mL); , TNF- (50 ng/mL) and VEGF (30 ng/mL). *P<.05 vs TNF-–treated cells. CNP-LI indicates CNP-like immunoreactivity.

View this table: [in this window] [in a new window]   Table 1. Effects of VEGF and Various Agents on CNP Concentrations in Conditioned Media of Cultured Bovine ECs

The Table also shows the comparison of the action of VEGF and bFGF, another potent angiogenic factor, on CNP secretion. Although 20 ng/mL bFGF exerted a stimulatory effect on EC proliferation comparable to that of 10 ng/mL VEGF, bFGF significantly stimulated CNP secretion by threefold. VEGF (30 ng/mL) also suppressed endothelial CNP secretion stimulated by bFGF (20 ng/mL).

Fig 4 shows Northern blot analysis of CNP mRNA with 1 µg poly(A)⁺ RNA. CNP mRNA with a size of 1.2 kb was detected in vehicle-treated ECs, as we reported. Exposure to VEGF (40 ng/mL) for 9 hours suppressed CNP mRNA expression by 40%. GAPDH mRNA levels were essentially equivalent among different RNA samples.

View larger version (48K): [in this window] [in a new window]   Figure 4. Northern blot analysis of CNP mRNA in bovine ECs. Confluent bovine ECs were exposed to TNF- (40 ng/mL) (left lane) or TNF- (40 ng/mL) plus VEGF (40 ng/mL) (right lane) for 9 hours. Poly(A)+ RNA (1 µg) was used for analysis. C indicates control; V, VEGF-treated.

	Discussion

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The present study clearly demonstrated that CNP secretion from ECs is suppressed by VEGF, a potent endothelium-specific angiogenic factor, in a time- and dose-dependent manner at the concentrations examined (1 to 100 ng/mL). Previous studies reported that in humans the VEGF concentration in the pleural effusion associated with malignancy, the vitreous fluid in diabetic proliferative retinopathy, and the synovial fluid in rheumatoid arthritis is 1 to 500 ng/mL.^{33 34 35}

We have previously reported the stimulation of CNP secretion from ECs by various growth factors and cytokines, especially TGF-ß, TNF-, and interleukin-1. These factors are considered to play crucial roles in vascular remodeling and the regulation of vascular functions in atherosclerosis and many inflammatory processes, especially endotoxin shock.^{12 13 15} So far, however, no vasoactive substances have been demonstrated to suppress CNP secretion from ECs. In the present study, VEGF is shown to potently suppress the secretion of CNP from ECs. Since we observed EC proliferation by VEGF, the suppressive action of VEGF on CNP secretion was not considered to be due to the nonspecific cytotoxic action of VEGF. VEGF exerted the inhibitory effect on CNP secretion with similar potency when different stimuli were used to promote CNP secretion. We consider that the suppressive action of VEGF on CNP secretion depends on common signaling pathways of the stimulation of CNP secretion by these stimulatory agents. Recently, it was reported that VEGF promotes tyrosine phosphorylation of several signal transduction mediators that contain SH2 domains³⁶ and finally activates mitogen-activated protein kinases through flt-1 and KDR or through the interaction of these two receptors³⁷ ; however, postreceptor signal transduction of VEGF has not been clarified enough.³⁸ The mechanism by which VEGF inhibits endothelial CNP secretion requires further investigation.

Angiogenesis plays a pivotal role not only in wound healing and tumor progression but also in diabetic angiopathy, arteriosclerosis, and collateral formation of obstructive vascular diseases. VEGF is an angiogenic growth factor that binds to its specific cell surface receptors (flt-1 and Flk-1/KDR) and thereby induces proliferation and migration of ECs. In addition, it is reported that VEGF increases vascular permeability and is able to attract monocytes³⁹ and regulate the gene expression of tissue plasminogen activator and collagenase^{40 41 42} at higher doses. The present finding is the first evidence that VEGF can regulate the expression of endothelium-derived vasoactive peptide.

We and others have elucidated that natriuretic peptides can act not only as vasodilators but also as growth inhibitors of vascular SMCs.^{16 17 18 19 20} Since the ANP-B receptor is demonstrated to be not as abundant in ECs as in SMCs,²¹ endothelial CNP can act as a paracrine factor to induce relaxation and/or growth inhibition of vascular SMCs more prominently rather than as an autocrine factor to modulate endothelial proliferation and/or function. The proliferation and migration of vascular SMCs with subsequent intimal thickening are major events in the development of an atherosclerotic lesion and restenosis after angioplasty.⁴³ Several growth factors are thought to be involved in these processes. TGF-ß, a potent growth inhibitor of ECs and bifunctional growth regulator of vascular SMCs, is released from platelets and synthesized by vascular SMCs and ECs.^{44 45} In addition, TGF-ß has been shown to be abundant in neointima after vascular injury,⁴⁶ suggesting its significant role in vascular remodeling. We previously reported that TGF-ß potently stimulates CNP secretion.¹² The specific and potent stimulatory action of TGF-ß on CNP secretion raises the possibility that the action of TGF-ß on vascular growth may be due in part to the augmentation of CNP secretion. Similarly, although VEGF can directly exert growth-stimulatory action solely on ECs through its specific receptors expressed in ECs, the present study suggests that locally generated VEGF could act as a paracrine factor to regulate SMC proliferation indirectly through the regulation of CNP secretion from ECs. In vascular injury lesion, regeneration of ECs was reported to crucially determine the extent of vascular SMC proliferation.⁴⁷ The present findings, therefore, deserve further investigation in the proliferative vascular lesion.

It has been reported that interleukin-1, TGF-ß, which we demonstrated to stimulate CNP secretion from ECs, or both increase VEGF expression in SMCs.^{48 49} The present study demonstrated that VEGF suppresses augmented secretion of CNP from ECs treated with TGF-ß. It is thus possible that TGF-ß modulates CNP secretion in an opposite way, ie, by direct stimulation and indirect inhibition through VEGF stimulation. The actual effect of TGF-ß in the interaction of ECs and SMCs in vivo requires further investigation.

In conclusion, the present study demonstrates a novel action of VEGF on the regulation of CNP secretion from ECs. The findings suggest that VEGF can regulate vascular tone and growth through suppression of the secretion of CNP, which is an endothelium-derived vasorelaxing and growth-inhibitory peptide.

	Selected Abbreviations and Acronyms

 

ANP = atrial natriuretic peptide bFGF = basic fibroblast growth factor BNP = brain natriuretic peptide CNP = C-type natriuretic peptide DMEM = Dulbecco's modified Eagle's medium EC = endothelial cell FCS = fetal calf serum SMC = smooth muscle cell TGF-ß = transforming growth factor–ß TNF- = tumor necrosis factor– VEGF = vascular endothelial growth factor

	Acknowledgments

 
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; Smoking Research Foundation; Yamanouchi Foundation for Research on Metabolic Disorders; Salt Science Research Foundation; Uehara Memorial Foundation; and Japanese Society for Cardiovascular Diseases. We thank Hisayo Kitoh, Mihoko Shida, and Yuko Mori for their excellent secretarial assistance.

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