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Cardiovascular Research 2003 57(1):232-237; doi:10.1016/S0008-6363(02)00612-0
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Copyright © 2003, European Society of Cardiology

Adrenomedullin augments the release and production of tissue factor pathway inhibitor in human aortic endothelial cells

Kousuke Marutsuka*, Kinta Hatakeyama, Atsushi Yamashita, Yuichiro Sato, Akinobu Sumiyoshi and Yujiro Asada

First Department of Pathology, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan

* Corresponding author. Tel.: +81-985-852-810; fax: +81-985-857-614 kmaru{at}post.miyazaki-med.ac.jp

Received 27 March 2002; accepted 7 August 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: Adrenomedullin (AM) is a hypotensive and vasodilative peptide. It has been reported that AM plays several biological roles in cardiovascular and endocrine systems, however, effects on the blood coagulation system have not been examined. In this study, we examined its effect on tissue factor pathway inhibitor (TFPI), which is a potent inhibitor of tissue factor/factor VIIa complex-induced coagulation cascade, and is synthesized and constitutively secreted by endothelial cells (ECs). The aim of this study was to elucidate the effects of AM on release and production of TFPI in ECs. Methods: Cultured human aortic ECs were incubated with AM (10–14–10–6 M), and the antigen levels and activity of the free and total form of TFPI after AM exposure were measured in culture media using ELISA. Furthermore, receptor interaction and cellular signaling of AM were investigated. Results: AM augmented TFPI release and production in a dose-dependent manner. The effect on TFPI production was inhibited by AM receptor antagonist (AM22–52), the monoclonal antibody against C-terminal region of AM, MAPKK inhibitor, and cAMP antagonist. Conclusion: These findings indicate that AM might play an important role in the modulation of anticoagulant properties in blood circulation.

KEYWORDS Anticoagulants; Arteries; Cell culture/isolation; Endothelial function; Hemostasis


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Adrenomedullin (AM) is a hypotensive and vasorelaxing peptide isolated from human pheochromocytoma tissue [1]. The peptide consists of 52 amino acids with an intramolecular disulfide bond forming a ring structure of six residues, and is widely distributed in a variety of tissues and organs, especially in cardiovascular and endocrine tissues [2–4]. Endothelial cells (ECs), smooth muscle cells, and cardiac myocytes are considered to be the main sources of AM in the cardiovascular system. AM has multifunctional biological activities, such as vasorelaxation, platelet cAMP increase, diuretic action, inhibition of aldosteron production, etc. [5], however, few reports have addressed its effect on hemostasis [6,7].

Blood coagulation is initiated by the binding of plasma protease factor VII/VIIa (FVIIa) to tissue factor (TF). The most physiological inhibitor of the TF–FVIIa complex is tissue factor pathway inhibitor (TFPI), which is the Kunitz domain-type serine protease inhibitor. TFPI directly inhibits factor Xa (FXa) and induces a feedback inhibition of the TF–FVIIa catalytic complex [8,9]. The major source of TFPI is ECs. ECs constitutively synthesize and secrete TFPI, and secreted TFPI is exposed on the cell surface via glycocalyx. TFPI is released from the luminal surface of ECs after an injection of either unfractionated heparin or low-molecular-weight heparins [10,11].

Recent clinical studies have shown that the plasma levels of both AM and TFPI are increased in thrombotic disease, such as acute coronary syndrome, disseminated intravascular coagulation, and thrombosis associated with advanced cancers [5,8,9,12–17]. The evidence suggests certain correlations between these molecules in ECs. We therefore investigated the effects of AM on expression and release of TFPI in cultured human aortic ECs.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Reagents
Recombinant full-length adrenomedullin (AM1–52); synthetic peptides of AM fragments containing 1 to 25 (AM1–25), 22 to 52 amino acid sequence of AM (AM22–52), and 8 to 37 amino acid sequence of calcitonin-gene related peptide (CGRP8–37) were purchased from the Peptide Institute (Osaka, Japan). Monoclonal antibodies against synthetic human AM12–25 (a ring structure) and AM46–52 (a native amidated C-terminal) were kindly provided by Dr. K. Kitamura (Department of Internal Medicine, Miyazaki Medical College) [18]. PD98059 (mitogen-activated protein kinase kinase (MAPKK) inhibitor), Rp-isomer-8-bromo-cAMP (cAMP antagonist), calphostin C (protein kinase C (PKC) inhibitor), NG-nitro-L-arginine-methylester (L-NAME), cyclohexamide and actinomycin D were purchased from Calbiochem (La Jolla, CA, USA).

2.2 Cell culture
Human aortic endothelial cells (HAoECs) were purchased from Sanko Junyaku (lot. 6F0741, Tokyo, Japan). The cells were grown on 6-well culture plates (Becton Dickinson Labware, Oxnard, CA, USA) in EGM-2 growth medium (Sanko Junyaku), containing 2% fetal bovine serum, growth factors (hFGF-B, VEGF, IGF-1, and hEGF), and antibiotics. Cells during 4 to 6 passages were used in this study. The cells were incubated with serum-free medium supplemented with 0.2% bovine serum albumin (SFM) at least 24 h before experiments to produce quiescence. The assay was carried out with SFM. The number of viable HAoECs was counted by using Trypan-Blue dye exclusion test or colorimetric assay with TetraColor One (Seikagaku Co.).

2.3 Cell treatments
HAoECs in a confluent state were incubated by various concentrations of AM (10–14 to 10–6 M) up to 48 h, and the cells were also incubated with pretreatment of unfractionated heparin (10 U/ml, Chromogenix, Molndal, Sweden) for 1 h to detach TFPI from the cell surface. To study the dependence of TFPI expression by AM on new protein synthesis and RNA transcription, HAoECs were pretreated with cyclohexamide (an inhibitor of protein synthesis) and actinomycin D (an inhibitor of RNA transcription) for 1 h or 24 h, respectively, before experiments with or without heparin treatment. To determine the active site of AM on TFPI expression, synthetic peptides of AM fragments (AM1–25, 22–52), CGRP8–37, and the two monoclonal antibodies against AM12–25 and AM46–52 were co-incubated with AM1–52 for 12 or 24 h with or without heparin pretreatment. Additionally, to examine the cellular signal transduction pathway, the heparin-pretreated cells were preincubated with PD98059 (5x10–9 to 10–5 M), calphostin C (10–9 to 10–6 M) or L-NAME (10–5 to 10–3 M) for 1 h at 37 °C and then incubated with AM for 24 h. The cells were incubated with both Rp-8-bromo-cAMP (1x10–7 to 10–3 M) and AM for 24 h at 37 °C.

2.4 Determinations of TFPI antigen and activity
TFPI antigen levels in the conditioned media (CM) were measured by one-step sandwich enzyme immunoassay by using total and free TFPI ELISA kits (Sanko Junyaku). TFPI antigen levels were expressed as ng/105 viable cells. TFPI activity was measured by a chromogenic assay using S-2222 (Chromogenix) and a rabbit polyclonal anti-TFPI antibody (kindly provided by Dr. Kamikubo, The Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan) as previously described [19].

2.5 Statistical analysis
Each data represents the mean±S.D. from two separate experiments in triplicate (n=6). Statistical analysis was performed by unpaired Student's t-test. Values of P<0.05 were accepted as significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Effect of AM on TFPI release and production
AM augmented dose-dependently the antigen levels of the free form of TFPI in CM at 24 h (Fig. 1). TFPI activity levels were also significantly increased by AM (Fig. 2). TFPI antigen level was increased 1 h after an addition of AM (10–12 or 10–6 M) in CM, and further increase was detected after 12 h (Fig. 3A). To determine whether the TFPI antigen is released from the cell surface or newly produced by HAoECs, the cells were pretreated with unfractionated heparin (10 U/ml) for 1 h to detach the pre-existing TFPI from the cell surfaces. In the study using the heparin-treated cells, TFPI antigen levels in CM were not increased by AM at 1, 3 and 6 h, but significantly increased after 12 h (Fig. 3B). These findings suggest that rapidly increased TFPI in the early phase (at 1, 3, 6 h) is derived from the cell surface binding type, and that of the late phase (>12 h) is newly produced by the HAoECs. The TFPI increase in the late phase, but not in the early phase, was completely inhibited by pretreatment of cyclohexamide (10 µg/ml) for 1 h or actinomycin D (10 µg/ml) for 24 h (Fig. 4), which supposed that TFPI production induced by AM needs new protein and mRNA synthesis.


Figure 1
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  		Fig. 1 Dose-dependent effect of AM on TFPI expression in CM of HAoECs. Confluent cultures of HAoECs were incubated with increasing concentrations of AM for 24 h. The CM was harvested and TFPI antigen levels were measured by free TFPI ELISA kit. The cells were trypsinized and viable cell number was counted with Trypan-Blue dye exclusion method. Results were expressed as mean±S.D. of ng/105 cells. *P<0.01 vs. control.

 

Figure 2
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  		Fig. 2 Dose-dependent effects of AM on TFPI activity in CM of HAoECs. TFPI activity was expressed as mean±S.D. of arbitrary units (AU)/105 cells. The inhibitory activity of 1 ng/ml human recombinant TFPI was defined as 1 AU of inhibitory activity. The reduced inhibitory activity by anti-TFPI polyclonal antibody is regarded as TFPI activity. *P<0.05 vs. control, **P<0.01 vs. control.

 

Figure 3
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  		Fig. 3 Time-course effect of AM on TFPI expression in CM of HAoECs. TFPI antigen levels in the CM of the cells (A) or heparin-pretreated cells (B) were measured. TFPI antigen levels were expressed as mean±S.D. of ng/105 cells. *P<0.05 vs. control, **P<0.01 vs. control (black bar, control; white bar, AM 10–12 M; gray bar, AM 10–6 M).

 

Figure 4
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  		Fig. 4 Inhibitory effects of cyclohexamide and actinomycin D on AM-induced TFPI expression. HAoECs were pretreated with cyclohexamide (10 µg/ml) for 1 h or actinomycin D (10 µg/ml) for 24 h, before experiments with or without heparin treatment. CM of the cells incubated with AM (10–6 M) for 3 h or 24 h were measured. TFPI antigen levels were expressed as mean±S.D. of ng/105 cells. *P<0.01 vs. AM at 24 h with or without heparin pretreatment. Heparin (–), experiment without heparin treatment; heparin (+), experiment with heparin pretreatment; CHX, cyclohexamide; ActD, actinomycin D.

 
3.2 Active site of AM on TFPI production
To determine the active sites of AM on TFPI production, we examined the effects of fragment peptides of AM (AM1–25 and AM22–52) on the heparin-pretreated HAoECs. TFPI production was significantly inhibited by AM22–52 (10–8 and 10–6 M), but not by AM1–25 (Fig. 5A). CGRP8–37 (10–12 to 10–4 M), which is also known as an antagonist for AM receptor, had no effect on TFPI production in this study. TFPI production was significantly inhibited by the monoclonal antibody against AM46–52 (a native amidated C-terminal), but not against AM12–25 (a ring structure) (Fig. 5B). While, in the experiment using the cells without heparin pretreatment, these agents or antibodies did not significantly affect TFPI concentrations in the medium at early phase (1 to 6 h) (data not shown).


Figure 5
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  		Fig. 5 Effects of AM antagonists (A) and specific antibodies (B) on AM action of TFPI production. Heparin-pretreated HAoECs were co-incubated with AM and the antagonists or antibodies. TFPI antigen levels were expressed as mean±S.D. of ng/105 cells. *P<0.05 vs. AM 10–6 M; anti-C, antibody against AM46–52 (C-terminal); anti-R, antibody against AM12–25 (ring structure).

 
3.3 MAPKK inhibitor and cAMP receptor antagonist inhibited TFPI production
As shown in Fig. 6, both PD98059 and Rp-cAMP at the optimal concentrations of 5x10–7 M and 1x10–3 M, respectively, inhibited TFPI production, but not calphostin C at 1x10–6 to 10–4 M. L-NAME treatment did not affect TFPI production.


Figure 6
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  		Fig. 6 Effects of inhibitors of second messengers on AM-induced TFPI production. HAoECs were pretreated with heparin and further pre-incubated with PD98059 or calphostin C for 1 h or L-NAME for 24 h and then incubated with AM. Rp-8-cAMP was co-incubated with AM after heparin pretreatment. TFPI antigen levels were expressed as mean±S.D. of ng/105 cells. *P<0.05 vs. AM 10–6 M.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The present study demonstrated that AM at a concentration of 10–12 M upregulated a release and production of TFPI from cultured HAoECs, and more significantly induced at 10–6 M. Plasma AM concentrations in normal subjects averaged 5.05±0.21x10–12 M, but ranged to 13.6 to 786.4x10–12 M in patients with septic shock [20]. In addition, local levels of AM in vascular tissues may be much higher than plasma levels. The mechanism of TFPI upregulation, therefore, would be mediated in physiological concentrations of AM.

There are some reports on the plasma levels of AM and TFPI in relation to disseminated intravascular coagulation and to other disease, such as acute coronary syndromes, renal diseases and cancer [5,8,9,12–17]. Patients with these disorders are generally in hypercoagulative condition, and frequently associated with thrombosis [8,9]. The evidence of AM-induced TFPI production and release from ECs indicates an indirect antithrombotic effect of AM. Sugano et al. [7] have recently reported that AM inhibited angiotensin-II-induced expression of TF and plasminogen activator inhibitor-1 in cultured ECs and we have also reported that AM reduced TF expression of ECs associated with apoptosis [21]. Taken together, AM could play an important role in the anticoagulant mechanism.

Cell-associated TFPI exists on the endothelial cell surface by binding to heparan sulfate proteoglycans (HSPG) [8]. Heparin-like glycosaminoglycan (GAG), Glypican 3, and lipoprotein receptor-related protein (LRP) have been raised as candidates of TFPI receptor on the cell surface, and TFPI binding to Glypican 3 and LRP is thought to be more specific than that to GAG, including HSPG [22–26]. The C-terminal region of TFPI, which is positively charged, mediates binding to both HSPG and LRP [22,26,27]. It is thought that heparin can displace TFPI from the binding site of GAG on the EC surface and into the blood stream in the form of heparin–TFPI complexes [22,27]. In this study, increase of TFPI in CM in the early phase suggests TFPI release from cell surface of HAoECs, because the TFPI increase vanished after heparin pretreatment. And no difference between measurements of a free form and total TFPI antigen levels was found, which also suggests TFPI in CM is originated from dissociation of cell-associated TFPI. We assumed that TFPI binding sites in HSPG on HAoECs were replaced by AM due to its strong cationic charge provided by its amino acid.

Identification of the AM receptor has been extensively studied. A family of receptor activity-modifying proteins (RAMPs 1 to 3) has recently been identified [28–30]. Associated with calcitonin receptor-like receptor (CRLR), RAMP2 or RAMP3 is proposed to be the specific receptor for AM, and RAMP1 appears to be the CGRP receptor [28]. Many following studies have supported these results [31,32] (reviewed in Refs. [33,34]). However, their binding specificity and characteristics are different regionally in organs and tissues [34–36]. Although AM22–52 and CGRP8–37 have been used as specific AM receptor antagonists, their antagonized responses were various [33–35]. In this study, the effect of AM on TFPI production was significantly reduced by AM22–52 and the monoclonal antibody against AM46–52. The evidence suggests an importance of the C-terminal region of AM on cell binding.

Previous reports demonstrated that AM showed an increase of cAMP, MAPK activity, endothelial cell nitric oxide synthase (eNOS) resulting in an increase of intracellular calcium, and also complicated interaction between them (reviewed in Ref. [34]). In this study, the AM-induced TFPI increase was significantly inhibited by MAP kinase kinase inhibitor (PD98059) and cAMP antagonists (Rp-isomer of 8-bromo-cAMP) but not by PKC inhibitor (calphostin C) or eNOS inhibitor (L-NAME). Thus, the effects of AM in the production of TFPI were mainly mediated by the cAMP pathway, which might induce MAPK and PKA pathways, however, the precious mechanisms of intracellular signaling of AM are still unclear.

In conclusion, AM induced TFPI release and production by HAoECs via cAMP-mediated pathways. AM might play an important role in the modulation of anticoagulant properties in normal and pathological states.

Time for primary review 19 days.


   Acknowledgements
 
The authors thank Dr. Kazuki Nabeshima, the Department of Pathology, Miyazaki Medical College Hospital, for his helpful discussion. This work was supported by the grants-in-aid from a Grant-in-Aid for Scientific Research on Priority Areas (B-2), Japan.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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