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Cardiovascular Research 2005 67(1):134-141; doi:10.1016/j.cardiores.2005.02.022
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Copyright © 2005, European Society of Cardiology

Troglitazone and 15-deoxy-{Delta}12,14-prostaglandin J2 inhibit shear-induced coupling factor 6 release in endothelial cells

Hirofumi Tomitaa, Tomohiro Osanaia, Tsutomu Tokib, Satoko Sasakia, Naotaka Maedaa, Reiichi Murakamia, Koji Magotad, Minoru Yasujimac and Ken Okumuraa,*

aThe Second Department of Internal Medicine, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562 Japan
bDepartment of Pediatrics, Hirosaki University School of Medicine, Hirosaki, 036-8562 Japan
cDepartment of Laboratory Medicine, Hirosaki University School of Medicine, Hirosaki, 036-8562 Japan
dDaiichi Suntory Biomedical Research Co., Ltd., Osaka, 618-8503 Japan

* Corresponding author. Tel.: +81 172 39 5057; fax: +81 172 35 9190. Email address: okumura{at}cc.hirosaki-u.ac.jp

Received 21 September 2004; revised 5 February 2005; accepted 24 February 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: We previously showed that mitochondrial coupling factor 6 (CF6), an endogenous inhibitor of prostacyclin synthesis and a vasoconstrictor, is present on the surface of human umbilical vein endothelial cells (HUVEC) and is released outside of the cells by shear stress. We investigated the intracellular signaling mechanism for shear-induced release of CF6 in HUVEC and the effects of troglitazone and 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2), both peroxisome proliferator-activated receptor (PPAR)-{gamma} ligands, on it.

Methods and results: The release and gene expression of CF6 in HUVEC were enhanced by shear stress at 25 dyn/cm2, measured by radioimmunoassay and real-time RT-PCR, respectively. The intracellular content of CF6 was decreased after exposure to shear stress at 25 dyn/cm2. Transfection experiments with deletional and mutational CF6 promoter constructs, and with dominant negative mutant I{kappa}B kinase {alpha} (K44M) demonstrated that shear-induced CF6 transcription was dependent on nuclear factor-kappa B (NF-{kappa}B) activation. Pretreatment with troglitazone or 15d-PGJ2 inhibited the shear-induced release and gene expression of CF6, whereas fenofibric acid, a PPAR-{alpha} ligand, had no influence on them. Western blot and immunostaining showed that troglitazone and 15d-PGJ2 inhibited the shear-induced, reactive oxygen species (ROS)-mediated activation of NF-{kappa}B at the level of I{kappa}B protein.

Conclusions: The shear-induced gene expression and release of CF6 in HUVEC are mediated by the ROS-related activation of NF-{kappa}B signaling pathway. Troglitazone and 15d-PGJ2 inhibit them at the I{kappa}B protein level.

KEYWORDS Coupling factor 6; Shear stress; Vascular endothelial cells; PPAR-{gamma} ligands; Nuclear factor-kappa B


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Mitochondrial ATP synthase is a multi-subunit membrane-bound enzyme that catalyzes the synthesis of ATP by utilizing a proton electrochemical gradient [1]. It consists of three domains, namely the extrinsic and intrinsic membrane domains (F1 and F0, respectively) joined by a stalk [1,2]. Coupling factor 6 (CF6) is one of the subunits in the stalk and an essential component for energy transduction [3]. We recently demonstrated that CF6 is present not only in the mitochondria but also on the surface of human vascular endothelial cells and is released outside of the cells by mechanical forces such as shear stress [4] and by tumor necrosis factor {alpha} (TNF-{alpha}) [5]. We further showed that CF6 is an endogenous inhibitor of prostacyclin synthesis in human vascular endothelial cells and functions as an endogenous vasoconstrictor peptide in rats [6,7]. More recently, we showed that circulating CF6 is elevated in human hypertension and modulated by salt intake presumably via reactive oxygen species (ROS) [8]. In addition, we showed that CF6 is a novel risk factor for ischemic heart disease in patients with end-stage renal disease [9]. Therefore, the demonstration of the shear stress-mediated signaling mechanism for this peptide release seems to be useful in further understanding its in vivo role and in developing a therapeutic strategy aimed at inhibiting the progression of hypertension and cardiovascular events.

Nuclear factor-kappa B (NF-{kappa}B), one of the most important transcription factors, is activated by a number of stimuli including TNF-{alpha} and shear stress [10–12]. These stimuli enhance the release of CF6 from vascular endothelial cells [4,5]. Therefore, we first investigated the involvement of NF-{kappa}B signaling pathway in the process of shear stress-induced CF6 release. On the other hand, thiazolidinediones (TZDs) such as troglitazone are high-affinity ligands for peroxisome proliferator-activated receptor (PPAR)-{gamma} and antidiabetic insulin-sensitizing drugs [13]. The prostaglandin D2 metabolite, 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2), is the first natural ligand for PPAR-{gamma} [14]. These PPAR-{gamma} ligands were shown to counteract NF-{kappa}B activation [15–17] and possess anti-atherosclerotic and anti-hypertensive properties [18,19]. Thus, we further investigated the effects of troglitazone and 15d-PGJ2 on the gene expression and release of CF6 under shear stress and the involvement of NF-{kappa}B signaling pathway in it.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Materials
HuMedia-EG2 kit was purchased from Kurabo Co., Ltd., Osaka, Japan. Troglitazone was kindly provided by Sankyo Co., Ltd., Tokyo, Japan. 15d-PGJ2 was purchased from Cayman Chemical, Ann Arbor, MI, USA. Fenofibric acid (Feno), a PPAR-{alpha} ligand, was from Grelan Co., Ltd., Tokyo, Japan. Sep-Pak C18 cartridge was purchased from Waters Chromatography Co., Milford, Massachusetts, USA. Expression vector (pcDNA3.1(+)) encoding the dominant negative mutant I{kappa}B kinase (IKK) {alpha} (K44M) was a generous gift from Tanabe Seiyaku Co., Ltd., Osaka, Japan [20]. All other reagents were of the finest grade commercially available (Sigma Chemical, St. Louis, MO, USA).

2.2. Cell culture and transfection
Human umbilical vein endothelial cells (HUVEC) were cultured in HuMedia supplemented with 2% fetal bovine serum, 10 ng/ml recombinant epidermal growth factor, 1 µg/ml hydrocortisone, 5 ng/ml recombinant fibroblast growth factor, and 10 µg/ml heparin under 5% CO2 at 37 °C. Cells were transfected with expression vector encoding the dominant negative mutant IKK {alpha} (K44M) or empty vector using Effectene Transfection Reagent (Qiagen GmbH, Hilden, Germany), and the transfection efficiency was approximately 50%. The transfected cells were further selected up to more than 90% by the medium containing 200 µg/ml of geneticin (Gibco, Grand Island, N.Y., USA) and maintained, as previously described [21].

2.3. Shear stress apparatus and protocol
The investigation conformed with the principles outlined in the Declaration of Helsinki. HUVEC plated on a 100-mm tissue culture dish were exposed to a fluid shear stress with the use of a cone-plate viscometer, as previously described [4]. HUVEC monolayers were exposed to laminar shear stress at 25 dyn/cm2 for 3 or 6 h with or without troglitazone, 15d-PGJ2 and Feno, except that one monolayer was kept in a static condition. The magnitude of shear stress used in this study is compatible with that of arterial shear stress, which is typically in the range of 20 to 40 dyn/cm2 and is further enhanced by increased cardiac output or hypertension [22]. The medium was taken for the measurement of CF6 release by radioimmunoassay (RIA), and the cells were used for the determination of the gene expression of CF6 by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) or the measurement of the intracellular content of CF6 by RIA. Viability of the cells, which were transfected with vectors or treated with PPAR ligands, was assessed by protein measurement and trypan blue staining with or without shear stress.

2.4. RIA of CF6
We established a specific RIA system for the measurement of human CF6, as previously described [4,7]. Briefly, RIA samples were loaded onto a Sep-Pak C18 cartridge and dissolved in RIA buffer (0.05 M sodium phosphate buffer at pH 7.4, containing 0.5% bovine serum albumin, 0.08 M NaCl, 0.025 M EDTA 2Na, and 0.05% NaN3). The standard recombinant CF6 or the unknown sample was incubated with anti-CF6 antiserum diluent for 24 h, and then the tracer solution was added. After incubation for 24 h, anti-rabbit IgG goat serum diluent containing 10% polyethylene glycol 6000 and rabbit IgG at 200 µg/ml were added. After standing for 1 h, the tubes were centrifuged and radioactivity of the precipitate was measured by gamma counter.

2.5. Real-time quantitative RT-PCR
Total RNA was extracted from HUVEC after shear stress using the TRIzol Reagent (Life Technologies, CA, USA). A two-step RT-PCR was carried out according to the protocol supplied with the TaqMan Gold RT-PCR kit (Applied Biosystems, CA, USA). Oligonucleotide primers and TaqMan probe for CF6 were designed using Primer Express version 1.5 (Applied Biosystems). The forward and reverse primers were 5'-TCTTCAGAGGCTCTTCAGGTTCTC-3' and 5'-GCCACTGCTGTAACACCAATGT-3', respectively. The TaqMan probe was 5'-TCATTCGGTCAGCCGTCTCAGTCCAT-3'. It was labeled at the 5'-end with a fluorescent reporter dye FAM (6-carboxy-fluorescein) and at the 3'-end with a quencher dye TAMRA (6-carboxy-tetramethylrhodamine). Oligonucleotide primers and TaqMan probe for human glyceraldehydes 3-phosphate dehydrogenase (GAPDH) were purchased from Applied Biosystems. The amplicon sizes of CF6 and GAPDH were 87 bp and 226 bp, respectively. The standard curves of CF6 and GAPDH were linear between 0.1 and 250 ng/µl total RNA. Values were averaged from duplicate data and normalized with the human GAPDH.

2.6. Immunostaining and fluorescence microscopy
HUVEC were preincubated with or without troglitazone at 8 µg/ml for 15 min. After exposure to shear stress at 25 dyn/cm2 for 30 min, the cells were fixed and stained with anti-NF-{kappa}B p65 antibody (Santa Cruz Biotechnology), as previously described [21].

2.7. Western blot analysis
HUVEC were exposed to shear stress at 25 dyn/cm2 for 30 min with or without troglitazone, 15d-PGJ2, N-acetylcysteine (NAC), and NG-nitro-L-arginine methyl ester (L-NAME), respectively. The cells were harvested, pelleted, and resuspended in ice-cold lysis buffer (25 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Triton X-100). Equal amount of protein (40 µg/lane) was applied to SDS-polyacrylamide gel electrophoresis (5–20% gradient gel). Protein was transferred to a PVDF membrane (BioRad Laboratories). After blocking for 1 h, the membranes were incubated with anti-I{kappa}B{alpha} or anti-GAPDH antibodies (Santa Cruz Biotechnology) at 4 °C overnight and followed by 1 h incubation with the secondary antibody. Immunoreactive bands were detected by amplified alkaline phosphatase immunoblot kits (BioRad Laboratories) or ECL plus detection system (Amersham Pharmacia Biotech).

2.8. Luciferase assay and site-directed mutagenesis
HUVEC were transfected with CF6 promoter/reporter vectors containing or lacking NF{kappa}B element. A 3.1-kb fragment containing the 5'-flanking region of the CF6 promoter was amplified by PCR (LA PCR, Takara, Japan) and subcloned into the luciferase reporter vector (Picagene basic vector, Toyo Ink Mfg. Co., Ltd., Tokyo, Japan) (F1). A deletion series of constructs lacking NF-{kappa}B element was generated by restriction enzyme digestion and subcloned into the luciferase reporter vector as shown in Fig. 3 (F2 and F3). The mutant CF6 promoter constructs, in which the binding site for NF-{kappa}B was abolished, were generated, as previously described [23]. A pair of primers containing a double point mutation (underlined) was designed in inverted tail-to-tail directions to amplify the mutated CF6 promoter/reporter vectors: 5'-TAACCTAACATAAACACTAAAATAGG-3' and 5'-GCCCCAGGTGTCCCTGCTGATA-3'. The amplified products were blunted and self-ligated to make the mutated constructs. The mutations were confirmed by sequencing. The pcDNAI/Neo-β-galactosidase plasmid was also cotransfected to monitor the transfection efficiency. The transfected cells were incubated at 37 °C for 24 h, and then exposed to shear stress at 25 dyn/cm2 for 3 h. The cells were harvested, and cell lysates were assayed with Picagene Luciferase Assay System (Toyo Ink) and Galacto-Light Plus System (Applied Biosystems) for the detection of β-galactosidase, as recommended by the manufacturer's instructions. The luciferase activity was normalized with that of β-galactosidase.


Figure 3
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  		Fig. 3 Effect of shear stress on CF6 promoter activity. Cells were cotransfected with deletional or mutational CF6 promoter/luciferase reporter vector and a β-galactosidase expression vector. Twenty-four hours after transfection, the cells were exposed to shear stress at 25 dyn/cm2 for 3 h. One monolayer was kept in a static condition. Luciferase activity normalized by that of β-galactosidase was expressed in fold increase over static condition. A double point mutation in NF-{kappa}B binding site is underlined. *P<0.05 vs. static condition, n=3. Ex1: exon 1; Luc: luciferase gene.

 
2.9. Data analysis
All data are shown as mean ± S.E.M. An unpaired t test for comparison of two variables and one-way or two-way ANOVA followed by Fisher's protected least significant difference test were used for statistical analysis. Differences were considered significant when P values were <0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Effects of shear stress on the release, gene expression, and intracellular content of CF6
As shown in Fig. 1, exposure of HUVEC to shear stress at 25 dyn/cm2 significantly enhanced the release of CF6 to the levels of 17.8 ± 3.0 ng/dish at 3 h and 62.4 ± 8.3 ng/dish at 6 h compared with a static condition (P<0.05 by two-way ANOVA). The intracellular content of CF6 was significantly decreased from 21.1 ± 2.9 ng/dish to 10.5 ± 0.6 ng/dish at 3 h after shear stress at 25 dyn/cm2 (P<0.05, n=3).


Figure 1
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  		Fig. 1 Effect of shear stress on the release of CF6 into the medium. One monolayer was kept in a static condition (static). *P<0.05 vs. initial time and **P<0.05 vs. 3 h after shear stress at 25 dyn/cm2 by one-way ANOVA followed by Fisher's PLSD, n=6.

 
Fig. 2A and B illustrate the representative bands and amplification curves of CF6 and GAPDH mRNA in real-time quantitative RT-PCR, respectively. Exposure of HUVEC to shear stress at 25 dyn/cm2 for 3 h significantly increased the ratio of CF6 to GAPDH mRNA by 2.5 ± 0.3-fold (Fig. 2C), and the similar increase in its ratio (2.2 ± 0.5 fold) continued at 6 h (P<0.05 vs. static, n=3). Exposure to shear stress at 15 dyn/cm2 for 3 h also increased the ratio of CF6 to GAPDH mRNA by 1.8 ± 0.7 fold (P<0.05 vs. static, n=3).


Figure 2
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  		Fig. 2 Effect of shear stress on the gene expression of CF6. (A) Representative bands for CF6 and GAPDH in real-time quantitative RT-PCR (40 cycles). (B) Representative amplification curves for CF6 and GAPDH. (C) Gene expression of CF6 normalized by the human GAPDH, n=4.

 
3.2. CF6 promoter activity after shear stress
To investigate the involvement of NF-{kappa}B in the CF6 promoter activity after shear stress, luciferase assay using a deletion series of CF6 promoter constructs was performed. Concerning the shear stress-responsive transcriptional factors, NF-{kappa}B, activator protein-1 (AP-1), early growth response-1, and specificity protein-1 (SP-1) have been reported [11,24]. As shown in Fig. 3, 5 AP-1 and 2 SP-1 binding sites and one NF-{kappa}B site were identified in the CF6 promoter (F1). Luciferase activity in the cells transfected with CF6 promoter constructs containing NF-{kappa}B element (F1) was increased by 2.4 ± 0.7-fold compared with static control after shear stress at 25 dyn/cm2 for 3 h, whereas it was unchanged in the cells with constructs lacking NF-{kappa}B element (F2 and F3) after shear stress. The double point mutation of NF-{kappa}B abolished the shear stress-induced increase in the luciferase activity of the F1 construct.

3.3. Analysis using dominant negative mutant IKK{alpha} (K44M) expression vector
To determine whether activation of NF-{kappa}B is responsible for the enhanced gene expression of CF6 by shear stress, HUVEC were transfected with dominant negative mutant IKK{alpha} (K44M) expression vector or empty expression vector [12,20]. As shown in Fig. 4A, in the cells transfected with empty expression vector, the release of CF6 was significantly increased from the levels of 1.5 ± 0.2 ng/dish to 19.4 ± 6.9 ng/dish after a 3-h exposure to shear stress at 25 dyn/cm2, whereas in the cells transfected with dominant negative mutant IKK{alpha} (K44M) expression vector, it was suppressed to the level of 4.0 ± 1.0 ng/dish after shear stress. Similarly, the gene expression of CF6 was enhanced by 2.0 ± 0.2-fold after a 3-h exposure to shear stress at 25 dyn/cm2 in the cells transfected with empty expression vector, whereas it was blocked in the dominant negative mutant transfected-cells (Fig. 4B).


Figure 4
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  		Fig. 4 Effect of transfection of dominant negative mutant IKK{alpha} (K44M) expression vector. Cells were transfected with either empty expression vector (empty) or dominant negative mutant IKK{alpha} (K44M) expression vector (mutant). The cells were exposed to shear stress at 25 dyn/cm2 for 3 h. (A) Release of CF6, n=4-6. (B) Gene expression of CF6, n=3. *P<0.05 vs. static by one-way ANOVA followed by Fisher's PLSD.

 
3.4. Effects of troglitazone, 15d-PGJ2, and Feno on the release, gene expression, and intracellular content of CF6
As shown in Fig. 5A, pretreatment of HUVEC with troglitazone at ≥ 0.8 µM or 15d-PGJ2 at ≥ 0.3 µM suppressed the increase in CF6 release after a 3-h exposure to shear stress at 25 dyn/cm2 in a dose-dependent manner. It also reversed the decrease in the intracellular content of CF6 after shear stress at 25 dyn/cm2 for 3 h (troglitazone at 8 µM: 21.8 ± 2.1 ng/dish, 15d-PGJ2 at 3 µM: 19.4 ± 4.4 ng/dish, n=4). In contrast, Feno at 300 µM, a PPAR-{alpha} ligand, had no influence on CF6 release. Under these conditions, trypan blue-positive cells were undetectable and protein contents were unchanged. As shown in Fig. 5B, pretreatment with troglitazone at 8 µM or 15d-PGJ2 at 3 µM blocked the shear stress-induced increase in the gene expression of CF6, whereas Feno at 300 µM did not affect it. Pretreatment with troglitazone at 8 µM inhibited the shear stress-induced increase in the luciferase activity in the cells transfected with F1 constructs.


Figure 5
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  		Fig. 5 Effects of troglitazone, 15d-PGJ2, and PPAR-{alpha} ligand on the shear stress-induced release and gene expression of CF6. Cells were pretreated with or without each agent, and then exposed to shear stress at 25 dyn/cm2 for 3 h. (A) Release of CF6, n=3-5. (B) Gene expression of CF6, n=3–4. *P<0.05 vs. control (no agent) by one-way ANOVA followed by Fisher's PLSD. Feno: fenofibric acid.

 
3.5. Immunostaining of NF-{kappa}B p65 protein
As shown in Fig. 6A, NF-{kappa}B p65 protein was stained in the cytoplasm in a static condition (a). Exposure to shear stress at 25 dyn/cm2 for 30 min induced translocation of NF-{kappa}B p65 protein into the nucleus (b). Pretreatment with troglitazone at 8 µM blocked the shear stress-induced translocation of NF-{kappa}B p65 protein into the nucleus (c). Antibody specificity was verified by the absence of NF-{kappa}B immunostaining in control experiments in which nonimmune serum was used instead of the primary antibody.


Figure 6
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  		Fig. 6 Effects of troglitazone, 15d-PGJ2, NAC, and L-NAME on the shear stress-induced NF-{kappa}B activation. (A) Immunostaining of NF-{kappa}B p65 subunit. (a) static condition, (b) shear stress at 25 dyn/cm2 for 30 min without troglitazone, and (c) that with troglitazone at 8 µM. (B) Western blot for I{kappa}B{alpha} and GAPDH proteins under shear stress at 25 dyn/cm2 for 30 min with or without PPAR-{gamma} ligands. (C) that with NAC or L-NAME.

 
3.6. Western blot of I{kappa}B{alpha} protein
To further elucidate the mechanism by which troglitazone inhibits NF-{kappa}B signaling pathway, the expression of its inhibitory protein I{kappa}B{alpha} was examined by Western blot analysis. As shown in Fig. 6B, the immunoreactive band for I{kappa}B{alpha} protein was detected in a static condition. Exposure of HUVEC to shear stress at 25 dyn/cm2 for 30 min induced degradation of I{kappa}B{alpha} protein. Pretreatment with troglitazone at 8 µM or 15d-PGJ2 at 3 µM inhibited the shear stress-induced degradation of I{kappa}B{alpha} protein. To explore the upstream targets for shear stress-induced NF-{kappa}B activation, we evaluated the potential roles of ROS and nitric oxide using their antagonists, NAC and L-NAME, respectively. As shown in Fig. 6C, pretreatment with NAC, a glutathione precursor, at 30 mM inhibited the shear stress-induced degradation of I{kappa}B{alpha} protein, whereas L-NAME, a nitric oxide synthase inhibitor, at 100 µM had no influence on it.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
We previously demonstrated that CF6 is present on the surface of HUVEC and is released outside of the cells by mechanical stimulation such as shear stress. The major findings of the present study are as follows: shear stress enhanced gene expression and release of CF6 by activation of NF-{kappa}B signaling pathway via degradation of I{kappa}B protein. Troglitazone and 15d-PGJ2, but not PPAR-{alpha} ligand, inhibited the shear stress-induced increase in the gene expression and release of CF6 in HUVEC. The effects of troglitazone and 15d-PGJ2 were mediated by inhibition of NF-{kappa}B signaling pathway at the level of I{kappa}B protein.

4.1. Intracellular signaling mechanism for shear-induced CF6 release
CF6 is first synthesized as an immature form in the cytosol, then transported to the mitochondria by an import signal peptide present in the upstream of mature CF6 [25], and becomes an active form by the enzymatic deletion of the signal peptide. Because CF6 is stored in the mitochondria, cell damage could be responsible for the shear stress-induced release of CF6 into the medium. However, no cell damage was observed after exposure to shear stress. As to the gene expression of CF6, real-time quantitative RT-PCR revealed that it was enhanced after shear stress at 25 dyn/cm2 but only by 2-fold. It is noted that the increase in CF6 release in response to shear stress was greater than that in the gene expression of CF6. We therefore assessed the intracellular content of CF6 by RIA, and found that it was decreased after shear stress at 25 dyn/cm2 at 3 h and the degree of the decrease was almost equivalent to that of the increase in CF6 release. These suggest that shear stress at 25 dyn/cm2 stimulates not only the gene expression but also the secretion of CF6 from the intracellular stores, thereby leading to the remarkable increase in CF6 release. However, it is not excluded that CF6 mRNA stability may be altered by shear stress and the de novo protein synthesis following to the gene expression may be more contributive at the late phase of shear stress.

In the present study, we showed that exposure of HUVEC to shear stress at 25 dyn/cm2 for 30 min induced degradation of I{kappa}B{alpha} protein and led to translocation NF-{kappa}B p65 subunit into the nucleus, suggesting that shear stress activates NF-{kappa}B signaling pathway. We further showed that by luciferase assay and transfection analysis, NF-{kappa}B plays a pivotal role in the shear stress-induced enhancement of gene expression of CF6. To elucidate the upstream targets for NF-{kappa}B activation in response to shear stress, we evaluated the potential roles of ROS and nitric oxide. We found that NAC, but not L-NAME, inhibited the shear stress-induced degradation of I{kappa}B{alpha} protein, suggesting that ROS may be involved in the process of NF-{kappa}B activation in response to shear stress. NF-{kappa}B is the main transcriptional activator for the shear stress-mediated synthesis of CF6. However, NF-{kappa}B is not specific to shear stress response but functions as an activator for inflammatory responses [26]. Being consistent with this concept, we previously showed that stimulation with a pro-inflammatory cytokine, TNF-{alpha}, enhanced CF6 release concomitant with the increase in its gene expression [5].

4.2. Role of NF-{kappa}B signaling pathway in the inhibitory effects of troglitazone and 15d-PGJ2
We next investigated the effects of troglitazone, 15d-PGJ2, both known as PPAR-{gamma} ligand, and Feno, a PPAR-{alpha} ligand, on the shear stress-induced release and gene expression of CF6 in HUVEC. We found that the release and gene expression were both inhibited by troglitazone and 15d-PGJ2. High concentrations of troglitazone and 15d-PGJ2 may also activate PPAR-{alpha}, but this is unlikely to explain our results because the concentrations of troglitazone and 15d-PGJ2 used were in a range compatible with no activation of PPAR-{alpha} [14]. Indeed, PPAR-{alpha} activation by Feno at 300 µM did not affect the shear stress-induced gene expression and release of CF6.

It has been shown that troglitazone and 15d-PGJ2 suppress NF-{kappa}B activation stimulated with various chemical mediators such as pro-inflammatory cytokines and lipopolysaccharide [15–17]. In the present study, we first provided evidence that both troglitazone and 15d-PGJ2 inhibited the shear stress-stimulated, ROS-mediated NF-{kappa}B activation presumably at the level of I{kappa}B protein in HUVEC. Thus, the inhibitory effect of PPAR-{gamma} ligands on CF6 release seems unlikely to be associated with the modulation of the NF-{kappa}B binding to a responsive element.

4.3. In vivo roles of CF6 and inhibitory effect of troglitazone and 15d-PGJ2
We recently demonstrated that the plasma concentration of CF6 is higher in spontaneously hypertensive rats at the hypertensive stage than in normotensive Wistar Kyoto rats, and that intravenous administration of anti-CF6 antibody causes a transient and rapid reduction in arterial blood pressure [7]. More recently, we showed that circulating CF6 is elevated in human hypertension and modulated by salt intake presumably via ROS [8]. We further showed that CF6 is a novel risk factor for ischemic heart disease in patients with end-stage renal disease [9]. Thus, the inhibitory effects of PPAR-{gamma} ligands may provide further understanding of the in vivo role of CF6 in these pathological situations. Previously it was reported that troglitazone lowers blood pressure in diabetic or obese insulin-resistant patients by unknown mechanisms [19,27]. The present results raise the possibility that a novel vasoconstrictor peptide may be involved in its mechanism.

Laminar flow at physiological levels induces the synthesis of prostacyclin and nitric oxide in endothelial cells [24,28], and it is considered to be anti-inflammatory. On the other hand, CF6 is also released in these conditions. Because CF6 is an endogenous inhibitor of prostacyclin synthesis, CF6 and prostacyclin might antagonistically regulate inflammatory responses and systemic vascular tone.

In conclusion, this report shows that the shear stress-induced increase in the gene expression and release of CF6 is mediated by activation of NF-{kappa}B signaling pathway at the level of I{kappa}B degradation, and troglitazone and 15d-PGJ2 inhibit it by blockade of the NF-{kappa}B signaling pathway. The inhibitory effects of troglitazone and 15d-PGJ2 would provide further understanding of the in vivo role of CF6 in cardiovascular disorders and a novel therapeutic strategy aimed at preventing hypertension and cardiovascular events. Further investigations are required to clarify the physiological and pathological roles of CF6, and curative effects of PPAR-{gamma} ligands in vascular system.


   Acknowledgements
 
We gratefully thank Shoji Tsutaya and Eriko Matsuda for excellent technical supports in real-time quantitative RT-PCR analysis. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture (C 13670686), and the Research Foundation for Community Medicine (Tokyo, Japan).


   Notes
 
Time for primary review 27 days


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

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