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Endocrinology Vol. 145, No. 7 3331-3337
Copyright © 2004 by The Endocrine Society

Antioxidant Effect of Adrenomedullin on Angiotensin II-Induced Reactive Oxygen Species Generation in Vascular Smooth Muscle Cells

Takanobu Yoshimoto, Nozomi Fukai, Ryuji Sato, Toru Sugiyama, Naoko Ozawa, Masayoshi Shichiri and Yukio Hirata

Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, Tokyo 113-8519, Japan

Address all correspondence and requests for reprints to: Takanobu Yoshimoto, M.D., Ph.D., Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8513, Japan. E-mail: tyoshimoto.cme{at}tmd.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent adrenomedullin (AM) gene-targeting studies have proposed a novel concept that AM plays a protective role against oxidative stress in vivo. The present study was undertaken to explore the underlying molecular mechanism of the putative antioxidant action of AM against angiotensin II (Ang II)induced reactive oxygen species (ROS) generation in rat vascular smooth muscle cells (VSMCs). Intracellular ROS levels were measured by dichlorofluoroscein fluorescence. Redox-sensitive c-Jun amino-terminal kinase (JNK) and ERK1/2 activation and gene expression induced by Ang II in VSMCs were also studied. AM dose-relatedly (10–8–10–7 M) inhibited intracellular ROS generation stimulated by Ang II (10–7 M), as mimicked by dibutyl-cAMP, the effect of which was inhibited by the pretreatment with N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride, a protein kinase A inhibitor, and calcitonin gene-related peptide(8–37), an AM/calcitonin gene-related peptide receptor antagonist. Ang II induced JNK and ERK1/2 activation via a redox-sensitive manner, whereas AM inhibited JNK, but not ERK1/2, activation by Ang II. Furthermore, AM inhibited Ang II-induced redox-sensitive gene expression (plasminogen activator inhibitor-1 and monocyte chemoattractant protein-1) in the same manner as N-acetyl-L-cysteine, a potent antioxidant. AM also inhibited Ang II-induced up-regulation of Nox1, a critical membrane-bound component of reduced nicotinamide adenine dinucleotide phosphate oxidase in VSMCs, in the same degree as N-acetyl-L-cysteine. Our study demonstrates for the first time that AM directly inhibits intracellular ROS generation via an AM receptor-mediated and c-AMP-protein kinase A-dependent mechanism in VSMCs and that AM with its potent antioxidant action inhibits redox-sensitive JNK activation and gene expression induced by Ang II. These data suggest that AM plays a protective role as an endogenous antioxidant in Ang II-induced vascular injury.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEVERAL LINES OF evidence have been accumulated that indicate that oxidative stress and reactive oxygen species (ROS) participate in the pathogenesis of cardiovascular diseases, including hypertension and atherosclerosis (1, 2). In response to growth factors and cytokines, vascular smooth muscle cells (VSMCs) and endothelial cells generate intracellular ROS, such as superoxide ({bullet}O2–) and hydrogen peroxide (H2O2) (3, 4, 5, 6, 7). ROS is currently recognized as a modulator of intracellular redox state, which plays an important role as a second messenger in regulating signal transduction pathways and subsequent gene expression (3, 8, 9).

Angiotensin II (Ang II), a key peptide hormone in the renin-angiotensin- aldosterone system, plays pivotal roles in the pathogenesis of cardiovascular remodeling and diseases. Ang II mediates its biological actions via highly complex intracellular signaling pathways (10, 11). It has recently been shown that many of these effects of Ang II are mediated by generation of ROS through the activation of vascular reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. For example, Ang II-induced VSMC hypertrophy is inhibited by blocking NADPH oxidase or scavenging intracellular ROS (12), and Ang II-induced ERK1/2 activation and subsequent gene expression in VSMCs is also inhibited by pretreatment with antioxidants (13, 14), suggesting its redox-sensitive signaling in VSMCs.

Adrenomedullin (AM), a potent vasodilator peptide originally isolated from human pheochromocytoma (15), has recently been shown to be abundantly expressed by and secreted from vascular endothelial cells and functions in an autocrine/paracrine fashion (16). AM has pleiotropic effects, including cell growth (17, 18), migration (19, 20), apoptosis (21), inflammation (22, 23), angiogenesis (24), and hormone secretion (25). Shimosawa et al. (26) have recently shown that AM-deficient mice generated by gene targeting resulted in perivascular inflammation in coronary artery and increases in systemic and local oxidative stress and reversal of increased urinary isoprostane excretion by exogenous AM supplementation. These data are consistent with the notion that AM may play a protective role against oxidative stress as an endogenous antioxidant in vivo. However, the underlying cellular mechanism and the mode of action of AM have not been clarified yet.

These observations led us to examine whether AM directly inhibits intracellular ROS generation stimulated by Ang II in VSMCs and, if so, to determine the underlying cellular mechanism responsible for its antioxidant effect, via redox-sensitive MAPK activation and gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Ang II, rat AM, and human calcitonin gene-related peptide (CGRP)(8–37) were purchased from Peptide Institute (Osaka, Japan). DMEM were obtained from Flow Laboratories (Irvine, Scotland, UK), and fetal bovine serum and calf serum were obtained from Cell Culture Laboratories (Cleveland, OH). Antiphospho ERK1/2 antibody, anti-ERK1/2 antibody, c-Jun-glutathione-S-transferase fusion protein, and antiphospho-c-Jun antibody were purchased from Cell Signaling Technology (Beverly, MA). N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H-89), dibutyl-cAMP, N-acetyl-L-cysteine (NAC), and diphenyleneiodonium (DPI) were from Sigma (St. Louis, MO). PCR primers were synthesized by JbioS Inc. (Saitama, Japan). The doses of Ang II, AM, and pharmacological inhibitors (DPI, H-89, and NAC) used without nonspecific effects in the present study were chosen from previous studies (5, 12, 17, 18, 19, 20, 21, 27, 28, 29).

Cell culture
Rat aortic VSMCs were prepared from the thoracic aorta of 6-wk-old male Sprague Dawley rats using the explant method and cultured in DMEM containing 10% fetal calf serum at 37 C in a humidified atmosphere of 95% air-5% CO2 as previously described (30). Subcultured cells (passages 4–8) were starved with DMEM containing 0.2% calf serum for 24 h and used for subsequent experiments.

Measurement of intracellular ROS levels
Intracellular ROS generation was measured in live VSMCs by the modified method of Ushio-Fukai et al. (12). Briefly, starved cells grown on 35-mm glass-bottom dishes (Matsunami, Tokyo, Japan) were pretreated with or without test compounds for the indicated time periods and then stimulated with or without 10–7 M Ang II for 2 h. In some experiments, CGRP(8–37) or H-89 had been added 30 min before AM pretreatment. Then cells were incubated with 8 µM H2O2-sensitive fluorophore CM-H2DCFDA (Molecular Probe Inc., Eugene, OR) for 30 min at 37 C and imaged by inverted fluorescence microscopy (IX71; Olympus, Tokyo, Japan) equipped with a mercury lamp, an excitation filter (470–490 nm), a dichroic mirror (505 nm), and an emission filter (510–550 nm). The relative fluorescent intensities were recorded and analyzed using a charge-coupled device camera (CoolSNAP HQ; Nippon Roper, Chiba, Japan) with image analysis system (MetaMorph; Nippon Roper).

Western blot analysis for phospho ERK1/2
Western blot analysis was performed as described previously (31). Briefly, 10 µg of the cell lysates were subjected to SDS-PAGE and Western blot analysis with rabbit polyclonal antiphospho ERK1/2 or anti-ERK1/2 antibody (Cell Signaling Technology). After incubation with peroxide-conjugated donkey antirabbit antibody (Amersham, Aylesbury, UK), the proteins were detected using an enhanced chemiluminescence Western blotting detection kit (Amersham).

Measurement of c-Jun amino-terminal kinase (JNK) activity
JNK activity was measured by a solid-phase kinase assay using a stress-activated protein kinase/JNK assay kit (Cell Signaling Technology). Briefly, 250 µg of the cell lysate was incubated with glutathione-S-transferase-c-Jun(1–79) fusion protein beads at 4 C overnight. Complexes were collected, washed, and then incubated with 50 µl of kinase buffer containing 100 µM ATP at 30 C for 30 min. The reaction was terminated with 5x Laemmli sample buffer, and samples were subjected to SDS-PAGE and Western blot analysis, as described above, with phospho-c-Jun antibody to detect c-Jun phosphorylation by JNK.

Quantification of mRNA
Total RNA extraction from VSMCs and first-strand cDNA synthesis were performed as described previously (32). Rat plasminogen activator inhibitor-1 (PAI-1) and NADPH oxidase 1 isoform (Nox1) mRNA levels were quantified with real-time quantitative RT-PCR using fluorescent SYBR Green technology (LightCycler; Roche Molecular Biochemicals, Mannheim, Germany), as previously described (20, 32). PCR primers used for amplification are listed in Table 1Go. The mRNAs for rat monocyte chemoattractant protein-1 (MCP-1) and acid ribosomal phospho protein P0 (ARPP P0), a house-keeping gene, were quantitated by TaqMan fluorescence methods, as described previously (32), except using QuantiTect Probe PCR Kit (Qiagen, Valencia, CA) and LightCycler (Roche). The mRNA levels of the target sequence were normalized by those of ARPP P0, which was used as an endogenous internal control; the relative levels of each mRNA to that of ARPP P0 were calculated as shown in each figure.


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  		TABLE 1. Sequences of the PCR primers for real-time RT-PCR

 
Statistical analysis
Data were expressed as mean ± SEM. Differences between groups were examined for statistical significance using either ANOVA with Dunn’s post hoc test or the unpaired t test, if they were appropriate. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AM inhibits intracellular ROS generation stimulated by Ang II
We first examined the effect of AM on ROS generation stimulated by Ang II in VSMCs by measurement of dichlorofluoroscein (DCF) fluorescence by microfluorometry. Treatment with Ang II (10–7 M) for 2 h caused a greater increase (2.6-fold) of DCF fluorescence than in untreated cells (Fig. 1Go, A, B, and F), the effect of which was suppressed after pretreatment with DPI (10–5 M), an inhibitor of flavin-containing oxidases (Fig. 1Go, C and F). Pretreatment with AM induced dose-related (10–8–10–7 M) inhibition of the Ang II-stimulated ROS generation (Fig. 1Go, D–F). These data suggest that AM exerts its antioxidant effect by inhibiting intracellular ROS generation stimulated by Ang II in VSMCs.



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  		FIG. 1. AM inhibits intracellular ROS generation stimulated by Ang II in VSMCs. VSMCs pretreated with or without AM (10–7 or 10–8 M) or DPI (10–5 M) for 1 h were stimulated with Ang II (10–7 M) for 2 h. ROS generation was detected by DCF fluorescence using microfluorometry. A–E, Representative microscopic images of DCF fluorescence (magnification, x400). F, Quantified data in each experimental group. The data obtained from more than 100 cells from three independent experiments were expressed as fold increase compared with control (CTR). *, P < 0.05 vs. control; {dagger}, P < 0.05 vs. Ang II.

 
AM inhibits intracellular ROS generation via AM receptor-mediated and cAMP-protein kinase A (PKA)- dependent pathway
To determine whether the antioxidant effect of AM is mediated via a specific AM receptor-mediated and cAMP-PKA-dependent pathway, we tested the effects of receptor antagonist and cAMP-related compounds (Fig. 2Go).



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  		FIG. 2. AM inhibits intracellular ROS generation via receptor-mediated and cAMP-dependent pathway. VSMCs treated with or without AM (10–7 M), DPI (10–5 M), dibutyl-cAMP (10–3 M), H-89 (10–5 M), or CGRP(8–37) (3 x 10–5 M) for 0.5–1 h were stimulated with or without Ang II (10–7 M) for 2 h. In some experiments, CGRP(8–37) or H-89 was applied 30 min before the AM pretreatment. Quantification of ROS levels by fluorescence using microfluorometry and calculation and plotting of data are the same as in Fig. 1Go. *, P < 0.05 vs. control (CTR); {dagger}, P < 0.05 vs. Ang II.

 
Pretreatment with 3 x 10–6 M CGRP(8–37), an AM/CGRP receptor antagonist, completely abolished the AM-induced inhibition of intracellular ROS generation stimulated by Ang II. Because the cAMP-PKA pathway is an important postreceptor signal transduction pathway of AM, we next examined the involvement of the cAMP-PKA pathway in the inhibitory mechanism of AM on ROS generation. Dibutyl-cAMP (10–3 M) significantly (P < 0.05) inhibited the ROS generation by Ang II to a similar extent of that of AM (10–7 M). By contrast, the inhibitory effect by AM on ROS generation was completely abolished by H-89 (10–5 M), a PKA inhibitor. Treatment with AM, CGRP(8–37), dibutyl-cAMP, or H-89 alone did not show any effect on intracellular ROS levels. These results are consistent with the notion that AM exerts its antioxidant effect via the AM receptor-mediated and cAMP-PKA-dependent pathway.

AM inhibits JNK, but not ERK1/2, activation stimulated by Ang II
It has been well recognized that ROS generation in response to various external stimuli, including Ang II, is linked to the activation of MAPKs (8, 13, 33, 34). Therefore, we reasoned that the antioxidant effect of AM might modulate Ang II-stimulated redox-sensitive MAPKs, such as JNK and ERK1/2, in VSMCs.

Because JNK activation by Ang II, as measured by the solid-phase kinase assay, was detectable as early as 5 min and peaked at 15 min (data not shown), we first examined the effects of AM and NAC, a potent antioxidant, on JNK activity at 15 min after Ang II (10–7 M) stimulation (Fig. 3Go). AM (10–7 M) significantly (P < 0.05) inhibited Ang II-induced JNK activation to a similar degree as that of NAC (10–2 M), suggesting that AM inhibits Ang II-stimulated JNK possibly via a redox-sensitive pathway. Treatment with AM or NAC alone did not show any effect on JNK activity (Fig. 3Go).



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  		FIG. 3. AM inhibits JNK activation stimulated by Ang II in VSMCs. VSMCs pretreated with or without AM (10–7 M) or NAC (10–2 M) for 30 min were stimulated with or without Ang II (10–7 M) for 15 min. Then cells were harvested and lysed, and the JNK activity was measured by JNK kinase assay using c-Jun as substrate. A, Representative blot is shown. B, The intensity of each band on the blot was quantified by densitometric scanning. The data were shown as fold increase compared with control (CTR) from the mean values from three independent experiments. *, P < 0.05 vs. control; {dagger}, P < 0.05 vs. Ang II.

 
Next, we examined the effect of AM on Ang II-stimulated ERK1/2 activation determined by immunoblotting using phospho-specific anti-ERK1/2 antibody (Fig. 4Go). AM failed to inhibit Ang II-induced ERK1/2 activation, whereas NAC completely inhibited the ERK1/2 activation by Ang II. Treatment with AM alone stimulated ERK1/2 activation as reported previously (18), whereas treatment with NAC alone did not show any effect on ERK1/2 activation (Fig. 4Go). Taken together, our results suggest that AM selectively inhibits JNK, but not ERK1/2, activation, both of which are similarly activated via a redox-sensitive pathway by Ang II.



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  		FIG. 4. AM does not affect ERK1/2 activation stimulated by Ang II in VSMCs. VSMCs pretreated with or without AM (10–7 M) or NAC (10–2 M) for 30 min were stimulated with or without Ang II (10–7 M) for 5 min. Then cells were harvested and lysed, and the ERK1/2 activation was measured by immunoblot using phospho-specific anti-ERK1/2 antibody. A, Representative blots with phospho-specific anti-ERK1/2 (top) and total anti-ERK1/2 antibody (bottom). B, The signal ratio of phospho ERK1/2 to total ERK1/2 was quantified by densitometric scanning. The data were calculated and plotted the same as in Fig. 3Go. *, P < 0.05 vs. control (CTR); {dagger}, P < 0.05 vs. Ang II.

 
AM inhibits PAI-1, MCP-1, and Nox1 up-regulation by Ang II via redox-sensitive pathway
Intracellular ROS induced by several extracellular stimuli has been known to be involved in the regulation of transcription and gene expression (8, 9, 14, 35, 36, 37). Ang II has been shown to stimulate mRNA expression of several redox-sensitive genes, such as PAI-1 and MCP-1, in VSMC (14, 38). Furthermore, it has recently been reported that Ang II induces gene expression of Nox1, a critical membrane-bound component of NADPH oxidase involved in vascular ROS generation (39, 40). These observations led us to reason that the antioxidant property of AM may be involved in the regulation of Ang II-induced gene expression in VSMCs.

Therefore, we examined whether AM affects Ang IIinduced PAI-1, MCP-1, and Nox1 gene expression in VSMCs using real-time RT-PCR. Time course experiments revealed that maximum mRNA induction of PAI-1, MCP-1, and Nox1 was similarly observed at 3 h after Ang II stimulation (data not shown). AM at 10–8 and 10–7 M significantly (P < 0.05) and comparably suppressed the expression of all three genes induced by Ang II to a similar degree as that by NAC (Fig. 5Go). It is of note that treatment with NAC alone significantly (P < 0.05) increased steady-state PAI-1 mRNA levels but not MCP-1 and Nox1 mRNA levels, whereas both AM and NAC were comparable to NAC alone in inhibiting the Ang II-induced mRNA expressions of all three genes. Treatment with AM alone, however, did not affect steady-state mRNA levels of any of these genes. These results suggest that AM, with its potent antioxidant effect, inhibits the Ang II-induced up-regulation of three redox-sensitive genes (PAI-1, MCP-1, and Nox1) in the same manner as NAC.



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  		FIG. 5. AM inhibits redox-sensitive gene expression stimulated by Ang II in VSMCs. VSMCs pretreated with or without AM (10–8 and 10–7 M) or NAC (10–2 M) for 1 h were stimulated with or without Ang II (10–7 M) for 3 h. Cells were harvested, and total RNA extraction and mRNA quantification for PAI-1, MCP-1, and Nox1 by real-time RT-PCR were performed as described in Materials and Methods. The data from five different experiments were calculated and plotted the same as in Fig. 3Go. *, P < 0.05 vs. control (CTR); {dagger}, P < 0.05 vs. Ang II.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates for the first time that AM decreases intracellular ROS generation stimulated by Ang II in VSMCs via AM receptor-mediated and c-AMP-PKAdependent pathway. Intracellular ROS generation stimulated by Ang II in vascular cells is considered to be mainly derived from the activation of NADPH oxidase (5, 12, 40). Superoxide anion generated by NADPH oxidase is promptly converted by superoxide dismutase to H2O2, a relatively stable molecule and a major determinant for intracellular redox state, which functions as a signaling molecule (41). The intracellular H2O2 is subsequently scavenged by catalase or glutathione peroxidase (41). Because DPI completely inhibited the increase in DCF fluorescence by Ang II in the present study, increased ROS generation by Ang II was largely derived from NADPH oxidase. The inhibitory effect of AM on the Ang II-stimulated ROS generation in VSMC as demonstrated in this study is consistent with the results of a recent study (42), which showed that AM gene transfer reduced NADPH oxidase activity in rat myocardial tissues after ischemia and reperfusion injury. Although NADPH oxidase is a possible molecular target of the antioxidant action by AM, it remains to be determined whether AM inhibits ROS generation via inhibition of NADPH oxidase activity and/or activation of the ROS scavenging system.

Intracellular ROS has been shown to function as a second messenger to activate a set of MAPK family members, such as ERK1/2, JNK, and p38 MAPK (8, 13, 33, 34). In the present study, the redox-sensitive JNK activation by Ang II was inhibited by AM, suggesting that the antioxidant action of AM is partly responsible for blockade of redox-sensitive signal transduction pathway. In contrast, the redox-sensitive ERK1/2 activation by Ang II was not affected by AM. Actually, we previously reported that AM itself stimulates ERK1/2 activation in VSMCs through the mechanism independent of cAMP pathway (18), as confirmed in the present study. In addition, others have shown that ERK1/2 activation can be activated by Ang II even in the presence of antioxidant in VSMC (34). Thus the present findings suggest that the pleiotropic AM actions on activation of ERK1/2 are independent of its antioxidant action.

In the present study, no additive effect of AM on ERK1/2 activation was observed in Ang II-treated VSMCs. We previously reported that both Ang II and AM could activate the proline-rich tyrosine kinase/c-Src protein tyrosine kinase pathway, thereby leading to activation of the p21ras/MAPK kinase 1/ERK1/2 cascade (43, 44). Thus ERK activation by Ang II and AM appears to share, at least in part, common pathways, which may explain the nonadditive effect of AM on Ang II-induced ERK activation as observed in the present study.

It is now well established that intracellular ROS plays a pivotal role in the regulation of gene expression under different redox states within cells (8, 9). In addition, it has been shown that gene expression of many proinflammatory and prothrombogenic molecules, such as PAI-1 and MCP-1, are regulated in a redox-sensitive manner (8, 9, 14, 36, 38, 45). In this study, we clearly demonstrated that AM inhibits the redox-sensitive gene (PAI-1 and MCP-1) expression induced by Ang II in a similar manner as NAC, a potent antioxidant. Shimosawa et al. (26) have shown that perivascular inflammation in response to Ang II and salt loading is more markedly induced in AM-deficient mice compared with that in wild-type mice. Thus it is possible to assume that AM with its antioxidant property may possess a vasculoprotective effect on Ang II-induced vascular injury.

In the present study, although both NAC and AM inhibited Ang II-induced PAI-1 gene expression with an almost similar antioxidant effect, NAC, but not AM, somehow induced PAI-1 gene expression. PAI-1 gene expression has been shown to be mediated via activator protein (AP)-1-dependent transcriptional activation (46). AP-1, a redoxsensitive transcription factor, is generally activated under various pro-oxidant conditions; however, certain antioxidants, such as pyrrolidine dithiocarbamate and NAC, have been shown to paradoxically enhance DNA binding with AP-1 in other cell types (47, 48). Thus the mechanism responsible for the inhibitory effects on PAI-1 gene expression by AM and NAC may differ from the mechanisms of other genes.

Nox1 is a member of the gp91 phox family, a critical membrane-bound component of NADPH oxidase in VSMCs (39, 40). The present study reveals for the first time that Nox1 up-regulation induced by Ang II is mediated via a redox-sensitive mechanism, the effect of which is blocked by AM in the same manner as NAC. It has been shown that Nox1 up-regulated by Ang II is largely responsible for the sustained superoxide production at the later phase (39, 40). Thus the inhibitory effect of AM on the Ang II-induced Nox1 up-regulation may account for the mechanism of its potent antioxidant action on the sustained and later phase of Ang II-stimulated superoxide generation.

In conclusion, the present study revealed for the first time one of the cellular mechanisms of the antioxidant effect of AM in VSMCs. AM directly inhibits intracellular ROS generation stimulated by Ang II via a AM receptor-mediated and c-AMP-PKA-dependent mechanism and also inhibits the Ang II-stimulated redox-sensitive signal transduction (JNK) and gene expression (PAI-1, MCP-1, and Nox1). Thus, the present in vitro study lends strong support to the contention that AM plays a vasculoprotective role in Ang II-induced vascular injury as a potent endogenous antioxidant, as suggested from previous in vivo studies (26, 42, 49). However, the pathophysiological significance of AM as an endogenous antioxidant remains to be determined under various oxidative stress-related cardiovascular disease states.


   Footnotes
 
This work was supported in part by Grant-in-Aid from the Ministry of Education, Science, Sports, Culture and Technology of Japan.

Abbreviations: AM, Adrenomedullin; Ang II, angiotensin II; AP, activator protein; ARPP P0, acid ribosomal phospho protein P0; CGRP, calcitonin gene-related peptide; DCF, dichlorofluoroscein; DPI, diphenyleneiodonium; H-89, N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride; JNK, c-Jun amino-terminal kinase; MCP-1, monocyte chemoattractant protein-1; NAC, N-acetyl-L-cysteine; NADPH, reduced nicotinamide adenine dinucleotide phosphate; Noxl, NADPH oxidase 1 isoform; PAI-1, plasminogen activator inhibitor-1; PKA, protein kinase A; ROS, reactive oxygen species; VSMC, vascular smooth muscle cell.

Received November 21, 2003.

Accepted for publication April 1, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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