Clinical and Experimental Pharmacology and Physiology (2005)32, 536–540
INHIBITION OF CYCLIC STRAIN-INDUCED ENDOTHELIN-1
SECRETION BY TETRAMETHYLPYRAZINE
Wei-Fung Bi,* Hung-Yu Yang,
†Ju-Chi Liu,
†Tzu-Hurng Cheng,
†Cheng-Hsien Chen,
†Chun-Ming Shih,* Heng Lin,* Tze-Che Wang,* Wei-Shiung Lian,
‡Jin-Jer Chen,
‡Heng-Cheng Chiu
§and Nen-Chung Chang*
*Department of Medicine, Taipei Medical University Hospital,
†Department of Medicine, Taipei Medical
University–Wan Fang Hospital,
‡Department of Internal Medicine, National Taiwan University Hospital and
National Taiwan University College of Medicine and
§Cathay General Hospital, Taipei, Taiwan ROC
SUMMARY
1. Chuanxiong is a Chinese herb that has been used widely in China to treat vascular disorders. 2,3,5,6-Tetramethylpyra-zine (TMP) is one of the major components purified from
chuanxiong. Many studies have demonstrated that TMP is
effective in the treatment of cardiovascular diseases. However, the mechanism of action by which TMP exerts relaxation in vascular vessels remains unclear.
2. Endothelin (ET)-1 is a potent vasopressor synthesised by endothelial cells both in culture and in vivo. The aims of the present study were to test the hypothesis that TMP may alter strain-induced ET-1 secretion and to identify the putative underlying signalling pathways in endothelial cells.
3. We showed that TMP inhibits strain-induced ET-1 secretion. 2,3,5,6-Tetramethylpyrazine also inhibits the strain-induced formation of reactive oxygen species (ROS) and phosphorylation of extracellular signal-regulated kinases (ERK) 1/2. Furthermore, pretreating cells with TMP or the anti-oxidant N-acetyl-cysteine decreased strain-induced increases in ET-1 secretion and ERK1/2 phosphorylation. Using a reporter gene assay, TMP and N-acetyl-cysteine were demonstrated to also attenuate the strain-induced activity of the activator protein-1 reporter.
4. In summary, we have demonstrated, for the first time, that TMP inhibits strain-induced ET-1 gene expression, in part by interfering with the ERK1/2 pathway via attenuation of ROS formation. Thus, the present study provides important new insights into the molecular pathways that may contribute to the proposed beneficial effects of TMP in the vascular system.
Key words: endothelial cells, endothelin-1, extracellular signal-regulated kinases 1/ 2, reactive oxygen species, strain, tetramethylpyrazine.
INTRODUCTION
Chuanxiong is a Chinese herb that has been used widely in China to treat vascular disorders. 2,3,5,6-Tetramethylpyrazine (TMP) is one of the major components purified from chuanxiong. Many studies have demonstrated that TMP is effective in the treatment of cardiovascular diseases. 2,3,5,6-Tetramethylpyrazine has been shown to increase coronary blood flow in dogs1 and to serve as a powerful vasodilator.2,3 In addition, TMP was demonstrated to produce antihypertensive effects in rats with portal hypertension.4 Direct relaxation of vessels by TMP has been demonstrated in vascular tissue and smooth muscle cells.2,5,6 However, the mechanism of action by which TMP exerts relaxation in vascular vessels remains unclear.
Among the earliest indications of vascular dysfunction in atherosclerosis is an impaired regulation of vasomotion, repre-senting disturbed homeostasis of vascular cells.7 Key regulators of vasomotor function are the vasodilator nitric oxide and the vaso-constrictor endothelin (ET)-1. Among the endogenous mediators of cardiovascular disorders, ET-1, a 21 amino acid peptide, is a primary antecedent in coronary heart disease.8–11 Such effects are mediated by extremely potent vasopressors and mitogenic responses to ET-1 in the vasculature.12,13 Results from preclinical studies in humans, as well as from animals studies, have shown that plasma ET-1 levels are consistently elevated in many spasm-related cardiovascular diseases13,14 and that antagonists of ET receptors can substantially alleviate complications of such diseases.14,15
Endothelin-1 was originally isolated from a culture of porcine endothelial cells.15 Endothelial cells are constantly under the influence of mechanical forces, including cyclic strain, as a consequence of vessel contraction and relaxation. Recently, numerous studies have shown that oxidative stress, represented by reactive oxygen species (ROS), is capable of significantly altering vascular function.16–18 Thus, oxidative stress appears to be causally linked to the pathogenesis of atherosclerosis.16–18 Previous studies have demonstrated that intracellular ROS levels are elevated in endothelial cells after cyclic strain treatment.19,20 One previous study demonstrated that intracellular ROS mediate cyclic strain-induced ET-1 expression via the Ras/Raf/extracellular signal-regulated kinases (ERK) 1/2 signalling pathway.21 However, no studies exist that address the effects of TMP on ET-1 gene
Correspondence: Heng Lin, Division of Cardiology, Department of Medicine, Taipei Medical University Hospital, Taipei, Taiwan.
Email: [email protected]
W-FB and N-CC contributed equally to this work.
Received 22 September 2004; revision 27 December 2004; accepted 25 January 2005.
TMP inhibits ET-1 gene expression 537
induction in vascular endothelial cells. The aim of the present study was to investigate the effect of TMP on strain-induced ET-1 gene expression and to identify signalling protein kinase cascades that may be responsible for the putative effect of TMP.
METHODS
Materials
Imubind ET-1 ELISA kits were purchased from Amersham-Pharmacia (Amersham, UK). 2,7-Dichlorofluorescin diacetate (DCFH-DA) was obtained from Serva (Heidelberg, Germany). Hydrogen peroxide (H2O2)
was purchased from Acros Organics (Pittsburgh, PA, USA). 2,3,5,6-Tetramethylpyrazine was purchased from Aldrich (Milwaukee, WI, USA).
N-acetyl-cysteine (NAC) and all other chemicals of reagent grade were obtained from Sigma (St Louis, MO, USA).
Endothelial cell culture
Human umbilical vein endothelial cells (HUVEC) were isolated from human umbilical cords, as described previously.21 All procedures involving
human samples were conducted according to the Guidelines for Animal and Human Experimentation of Taipei Medical University. The transformed human cell line ECV304 (ATCC CRL-1998) was purchased from the American Type Culture Collection (Bethesda, MD, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, penicillin (100 U/mL) and streptomycin (100 g/mL) before subculture.
In vitro
cyclic strain of cultured endothelial cells
Endothelial cells cultured on a flexible membrane base were subjected to cyclic strain produced by computer-controlled application of sinusoidal negative pressure, as described previously.21
Measurement of ET-1 concentrations
Endothelin-1 levels were measured in the culture medium using a commercial ELISA kit. Results were normalized to cellular protein content in all experiments and are expressed as a percentage relative to cells incubated with vehicle (dimethylsulphoxide).
Detection of intracellular ROS
Measurement of intracellular ROS formation in HUVEC was recorded by monitoring changes in diclorofluorescein (DCF) fluorescence, as described previously.21 A chemiluminescence assay of superoxide production was
performed, as described previously.19
Western blot analysis
Rabbit polyclonal anti-phospho-specific ERK1/2 antibody was purchased from New England Biolabs (Beverly, MA, USA). The anti-ERK antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Western blot analysis was performed as described previously.21
Fig. 1 2,3,5,6-Tetramethylpyrazine (TMP) inhibits strain-induced endo-thelin (ET)-1 secretion. Human umbilical vein endothelial cells were pretreated with TMP (0.1–100 mol/L) 30 min prior to strain treatment. After strain (20%) for 24 h, the culture media were collected and the concentrations of ET-1 analysed by enzyme immunoassay. Results are the meanSEM (n = 6). *P < 0.05 compared with unstrained control cells;
†P < 0.05 compared with strained cells (
ANOVA). C, control.
Fig. 2 Effects of 2,3,5,6-tetramethylpyrazine (TMP) on strain-increased reactive oxygen species (ROS) formation. (a) Effect of TMP (0.1– 100 mol/L) on strain-induced ROS generation. Strain-induced increases in intracellular ROS were revealed by fluorescent intensities of 2,7 -dichloro-fluorescin (DCF). (b) Effect of TMP (0.1–100 mol/L) on strain-induced superoxide formation. Human umbilical vein endothelial cells (HUVEC), after cyclic strain, were lysed, which was followed immediately by the superoxide assay using the lucigenin method. (c) Data for control (C) HUVEC or those treated with cyclic strain or H2O2 (25 mol/L) in the
presence of 100 mol/L TMP or 10 mmol/L N-acetyl-cysteine (NAC) for 1 h. Fluorescence intensities of cells are shown as the relative intensity of experimental groups compared with untreated control cells. Data are the meanSEM (n = 6). *P < 0.05 compared with control; †P < 0.05 compared
538 W-F Bi et al.
Luciferase assay
The ECV304 cells, plated on 3 cm diameter culture dishes, were transfected with the luciferase reporter construct possessing consensus activator protein-1 (AP-1) binding sites (AP-1-Luc; Stratgene, La Jolla, CA, USA) using the calcium phosphate method, as described previously.21 After
incubation for 24 h in 2% serum DMEM, ECV304 were cultured under different treatments with TMP and NAC for 48 h. The ECV304 cells were assayed for luciferase activity with a luciferase reporter assay kit (Strate-gene). The firefly luciferase activities at AP-1 transcriptional activity were normalized for transfection efficiency to the respective -galactosidase activity and expressed as relative activity to control.
Lactate dehydrogenase assay
Cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) in the culture medium spectrophotometrically. Lactate dehydrogenase release was standardized using a cell injury index defined as (A – B)/ (C – B) 100, where A is LDH activity in the test sample, B is LDH activity measured in media with no cells (0% control) and C is LDH
activity in samples from wells in which cells were lysed with Triton X-100 (100% control).
Statistical analysis
Data are presented as the meanSEM. Statistical analysis was performed using Student’s t-test and analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test using Prism version 3.00 for Windows (GraphPad Software, San Diego, CA, USA). P < 0.05 was considered statistically significant.
RESULTS
Effect of TMP on strain-induced ET-1 secretion in
endothelial cells
Human umbilical vein endothelial cells cultured on flexible membrane bases were subjected to deformation to produce an average strain of 20%. Endothelin-1 released into the culture media was measured. Human umbilical vein endothelial cells under cyclic strain for 24 h demonstrated a twofold increase in the secretion of ET-1 into the culture medium (Fig. 1) compared with unstrained endothelial cells. The effect of TMP on strain-induced increases in ET-1 secretion was then investigated. As shown in Fig. 1, treatment with cyclic strain for 24 h increased ET-1 secretion, but 1–100 mol/L TMP significantly inhibited this strain-induced increase in ET-1 secretion (Fig. 1). These data indicate that TMP inhibits strain-induced increases in ET-1 secretion by endothelial cells.
To verify that the TMP-mediated effects on vascular endothelial cells are not a consequence of cytotoxicity, we determined lactate dehydrogenase (LDH) activity as a parameter of necrosis in the supernatants of TMP (1–100 mol/L)- and strain-treated HUVEC. Consistent with light microscopic observations, LDH activity in the supernatants of treated and untreated cells remained unchanged (data not shown).
Fig. 3 Inhibitory effects of 2,3,5,6-tetramethylpyrazine (TMP) on strain-increased extracellular signal-regulated kinase (ERK)1/2 phosphorylation. (a) Effect of TMP (1–100 mol/L) on strain-activated ERK1/2 phosphory-lation. (b) Effect of TMP on strain- or H2O2-induced phosphorylation of
ERK1/2. Human umbilical vein endothelial cells were pre-incubated with either TMP (100 mol/L) or N-acetyl-cysteine (NAC; 10 mmol/L) for 30 min and stimulated with cyclic strain or H2O2 (25 mol/L) for 30 min.
Phosphorylation of ERK1/2 was detected by western blotting using anti-phospho-ERK1/2 antibody. Both TMP and NAC inhibited strain-induced activation of ERK1/2. Phosphorylation of ERK1/2 was detected and densitometric analyses were performed. (), degree of ERK1 phosphory-lation; (), degree of ERK2 phosphorylation. Data are the fold increase relative to control groups and show the meanSEM (n = 6). *P < 0.05 compared with control; †P < 0.05 compared with strain (or H
2O2) alone
(ANOVA). C, control; pERK, phosphorylated ERK.
Fig. 4 2,3,5,6-Tetramethylpyrazine (TMP) attenuates the strain-stimulated activator protein-1 (AP-1) reporter activity in endothelial cells. ECV304, transfected with AP-1-Luc, were incubated for 24 h with either no drug, 100 mol/L TMP or 10 mmol/L N-acetyl-cysteine (NAC) in the absence or presence of strain treatment or H2O2 (25 mol/L). Data are the
meanSEM for six experiments performed in triplicate. *P < 0.05 compared with untreated; †P < 0.05 compared with strain (or H
2O2) alone
TMP inhibits ET-1 gene expression 539
2,3,5,6-Tetramethylpyrazine inhibits strain-increased
ROS formation
Previous studies have shown that cyclic strain increases ROS formation in endothelial cells,19,20,22 which is involved in ET-1 induction.21 We next examined whether TMP prevents strain-increased ROS formation. Human umbilical vein endothelial cells were treated with TMP (0.1–100 mol/L) in the absence or pres-ence of strain treatment. The addition of TMP (1–100 mol/L) to cultured HUVEC significantly inhibited strain-induced ROS formation measured after strain treatment for 1 h (Fig. 2a,b). Pretreatment of cultured HUVEC with TMP (100 mol/L) or N-acetyl-cysteine (NAC; 10 mmol/L) also significantly inhibited strain- or H2O2-induced ROS formation (Fig. 2c). These findings clearly demonstrate that TMP inhibits strain-induced increases in intracellular ROS levels in endothelial cells.
2,3,5,6-Tetramethylpyrazine inhibits strain-activated
ERK1/2 phosphorylation in endothelial cells
To gain an insight into the mechanism of action of TMP, we examined whether TMP affects intracellular protein kinase signalling pathways. Given that the ERK1/2 signalling pathway is involved in strain-induced ET-1 expression,21 we investigated whether TMP inhibits the ERK1/2 pathway in strain-treated endothelial cells. We examined the phosphorylation of ERK1/2 in HUVEC exposed to TMP (1–100 mol/L) in the absence or pres-ence of strain treatment. As shown in Fig. 3, HUVEC exposure to strain treatment for 30 min rapidly activated the phosphory-lation of ERK1/2. However, treatment of HUVEC with 100 mol/L TMP resulted in significantly decreased strain-induced ERK1/2 phosphorylation. Moreover, treatment of HUVEC with 25 mol/L H2O2 resulted in increased ERK1/2 phosphory-lation (Fig. 3). Human umbilical vein endothelial cells pretreated with 100 mol/L TMP or NAC 10 mmol/L showed significantly decreased strain- or H2O2-induced ERK1/2 phosphorylation. These findings imply that TMP inhibits the strain-activated ERK1/2
signalling pathway via attenuation of ROS formation in endothelial cells.
2,3,5,6-Tetramethylpyrazine inhibits strain-induced
increases in AP-1 transcriptional activity in
endothelial cells
The effect of TMP on AP-1 activation, which is involved in ET-1 gene induction,21,23 was then evaluated. The effects of TMP on strain-induced AP-1 functional activity were assessed in a reporter gene assay. Both TMP (10 mol/L) and NAC (10 mmol/L) signifi-cantly attenuated strain- or H2O2-induced AP-1 reporter activation (Fig. 4). These results indicate that TMP inhibits strain-induced increases in AP-1 transcriptional activation.
2,3,5,6-Tetramethylpyrazine inhibits strain-induced
ET-1 secretion, which is compatible with an
anti-oxidant action
To further determine whether TMP affects strain-induced ET-1 gene expression via attenuation of ROS formation, the effects of TMP on ET-1 gene induction were examined under cyclic strain or H2O2 stimulation. As shown in Fig. 5, neither TMP nor NAC alone had any effect on basal ET-1 secretion. However, HUVEC treated with either strain or H2O2 (25 mol/L) for 24 h demonstrated significantly increased ET-1 secretion (Fig. 5). In the presence of TMP (or NAC), both strain- or H2O2-induced increases in ET-1 secretion were significantly inhibited. These data imply that TMP affects strain-induced ET-1 gene expression, which is compatible with an anti-oxidant action in endothelial cells.
DISCUSSION
The major new finding of the present study is that TMP inhibits strain-induced ET-1 secretion in endothelial cells. This is supported by the observations that TMP inhibits strain-induced ET-1 protein secretion by, in part, attenuation of ROS formation in endothelial cells. Previous studies have indicated that haemodynamic forces, including shear flow24 and pressure-induced strain,21,25 can stimu-late ET-1 gene expression. Recent studies provide evidence that ROS may act as second messengers in cells exposed to various stimuli.21,23 Previous studies have shown that cyclic strain treatment of endothelial cells can induce intracellular ROS generation.19,20,26 This ROS generation is sustained at an elevated level as long as the mechanical forces are maintained and returns to basal levels following the removal of the mechanical forces. Elevated ROS levels are involved in the release of ET-121 and this gene induction can be attenuated by anti-oxidant pretreatment of cells.
Zhang et al. reported that TMP scavenges ROS generation in human polymorphonuclear leucocytes and cultured neurons.27,28 The results of the present study further demonstrated that TMP reduced strain-induced ROS generation in the endothelial cells. In particular, it has been demonstrated that the activation of ERK1/2 is redox sensitive21,29,30 and that suppression of ROS inhibits strain-induced ET-1 gene expression.21 Thus, one possible explanation for the inhibitory effect of TMP on strain-induced ET-1 gene expres-sion may be its ability to attenuate ROS formation. In a previous study, deletion mapping revealed constructs containing 143 bp of the ET-1 5-flanking sequence allowing strain-induced
transcrip-Fig. 5 2,3,5,6-Tetramethylpyrazine (TMP) modulates strain-induced endothelin (ET)-1 secretion, which is compatible with an anti-oxidant action. 2,3,5,6-Tetramethylpyrazine modulates strain (or H2O2)-increased
ET-1 secretion. Some cells were pretreated with 10 mmol/L TMP or 100 mol/L N-acetyl-cysteine (NAC) for 30 min. Endothelial cells were then treated with cyclic strain or H2O2 (25 mol/L) for 24 h. Data are the
meanSEM (n = 6). *P < 0.05 compared with control; †P < 0.05 compared
540 W-F Bi et al.
tion and the presence of responsive elements for ROS- or strain-induced ET-1 expression located within 143 bp upstream of the transcription initiation site.21 Recently, we also found that activation of AP-1 is redox sensitive and may play a key role in ET-1 gene induction.23 The results of the present study indicate that TMP inhibits strain-induced AP-1 reporter activity. The inhibitory effect of TMP on strain-induced AP-1 transcriptional activation suggests that the attenuation of strain-induced ROS by TMP leads to inhibition of AP-1.
In conclusion, the data of the present study suggest that the TMP-induced suppression of cyclic strain-induced ET-1 expression can be considered as one of the mechanisms responsible for the protective effect of TMP in vascular vessels. The effects of TMP on endothelial cells observed in the present study (i.e. inhibition of ET-1 secretion, suppression of ROS formation and inhibition of ERK1/2 phosphorylation) all are compatible with its putative vasoprotective effect.
ACKNOWLEDGEMENT
This work was supported, in part, by Cathay General Hospital Grant, Taiwan ROC (93CGH-TMU-05).
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