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Endocrinology Vol. 141, No. 9 3120-3126
Copyright © 2000 by The Endocrine Society

ARTICLES

Involvement of Reactive Oxygen Species in the Activation of Tyrosine Kinase and Extracellular Signal-Regulated Kinase by Angiotensin II1

Gerald D. Frank, Satoru Eguchi, Tadashi Yamakawa, Shun-ichi Tanaka, Tadashi Inagami and Evangeline D. Motley

From the Department of Anatomy and Physiology (G.D.F., E.D.M.), Meharry Medical College, Nashville, Tennessee 37208; Department of Biochemistry (G.D.F., S.E., T.Y., T.I.), Vanderbilt University School of Medicine, Nashville, Tennessee 37232; and Neurobiology of Aging Laboratories (S-i.T.), Mt. Sinai School of Medicine, New York, New York 10029

Address all correspondence and requests for reprints to: Evangeline D. Motley, Department of Anatomy and Physiology, Meharry Medical College, Nashville, Tennessee 37208. E-mail: emotley{at}mmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Reactive oxygen species (ROS) have been proposed to mediate vascular hypertrophy induced by angiotensin II (Ang II). Recently, we and others have shown that growth-promoting signals by Ang II involve protein tyrosine kinase (PTK) and extracellular signal-regulated kinase (ERK). However, whether ROS contribute to the Ang II-induced PTK and/or ERK activation in vascular smooth muscle cells (VSMCs) remains largely unclear. Here, we have investigated the possible involvement of ROS in Ang II-induced PTK and ERK activation. In the presence of a NADH/NADPH oxidase inhibitor, diphenyleneiodonium (DPI) or an antioxidant, {alpha}-tocopherol, Ang II-induced protein tyrosine phosphorylation of two major proteins (p120, p70) and ERK activation were markedly reduced, whereas ERK activation by epidermal growth factor was unaffected. DPI also inhibited Ang II-induced H2O2 production and PTK activation. In this regard, H2O2 and a membrane permeable thiol-oxidizing agent, diamide, stimulated protein tyrosine phosphorylation of p120 and p70, and ERK activation in VSMCs. H2O2 also enhanced PTK activity. From these data, we conclude that ROS play a critical role in the Ang II-induced PTK and ERK activation in VSMCs, thereby contributing to vascular growth associated with enhanced Ang II activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
REDUCTION of oxygen by normal cellular metabolism leads to the production of reactive oxygen species (ROS) that include superoxide anion (O2.-), hydrogen peroxide (H2O2), and hydroxyl radical (1). These species are now believed to participate in a variety of cellular signaling mechanisms that transmit transcriptional/translational regulation, cell growth, differentiation, and apoptosis (2, 3). In this regard, extracellular signal-regulated kinases (ERK) and other members of the mitogen-activated protein kinase (MAPK) family gain an enhanced activity as a result of ROS stimulation (4, 5). In addition, several protein tyrosine kinases (PTK) are reported to be activated by ROS in various cell lines (6, 7).

Accumulating evidence indicates that ROS play an important role in cardiovascular diseases such as hypertension, atherosclerosis, and restenosis after angioplasty (8, 9). The major effector peptide of the renin-angiotensin system, angiotensin II (Ang II), has been shown to stimulate O2.- production by activating NADH/NADPH oxidase, which is an enzyme that appears to be a major source of O2.- production in vascular smooth muscle cells (VSMCs) (10). Moreover, it has been reported that ROS mediate Ang II-induced hypertrophy in VSMCs (11, 12). The Ang II type 1 (AT1) receptor (13, 14) not only mediates various Ang II-induced hemodynamic effects (15) but also promotes hypertrophy and/or hyperplasia in VSMCs (16, 17). The AT1 receptor activates phospholipase C, which results in the generation of two second messengers, inositol triphosphate and diacylglycerol that, in turn, mobilizes intracellular Ca2+ stores and activates protein kinase C, respectively (18). AT1 receptor stimulation also induces protein tyrosine phosphorylation and activates ERK leading to c-Fos and c-Jun expression in VSMCs (10, 19, 20, 21, 22). In this regard, both PTK and ERK appear to be indispensable for the protein synthesis induced by Ang II in VSMCs (23, 24). Recently, we and others have identified some key PTKs that mediate ERK activation by Ang II in VSMCs. Theses PTKs include both receptor [epidermal growth factor (EGF) receptor] and nonreceptor PTKs (c-Src, Pyk2) (20, 25, 26). Although recently ROS have been shown to induce ERK activation as well as c-Fos and c-Jun expression in VSMCs (27), their involvement in the growth-promoting signal of Ang II remains largely unclear.

In this study, we examined the role of ROS in the signaling pathway of Ang II-induced PTK and ERK activation in VSMCs. We demonstrated that the NADH/NADPH oxidase inhibitor, diphenyleneiodonium (DPI), and the antioxidant, {alpha}-tocopherol, inhibited the Ang II-induced protein tyrosine phosphorylation and subsequent ERK activation. DPI also inhibited Ang II-induced PTK activation. We further showed that H2O2 and diamide were able to stimulate protein tyrosine phosphorylation and ERK phosphorylation, and that H2O2 enhanced PTK activity in VSMCs. These results indicate that ROS, produced via Ang II-stimulated NADH/NADPH oxidase, play a critical role in the Ang II-induced growth-promoting signal in VSMCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and reagents
Ang II, {alpha}-tocopherol, DPI, and the PTK substrate poly-[Glu80-Tyr20] were purchased from Sigma (St. Louis, MO). The Takeda Pharmaceutical Company generously provided the AT1 antagonist, CV11974. Monoclonal antiphosphotyrosine antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal antibodies for Thr202 and Tyr204-phosphorylated ERK 1/2 were purchased from New England Biolabs, Inc. (Boston, MA). Polyclonal antibodies against ERK 2 and protein A/G agarose were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Peroxidase-linked antirabbit and mouse IgG and ECL reagent were obtained from Amersham Pharmacia Biotech (Piscataway, NJ).

Cell culture
VSMCs were prepared from the aorta of 12-week-old male Sprague Dawley rats (Charles River Laboratories, Inc. Breeding Laboratories) by the explant method as previously described (19). Subcultured cells from passages 3–15 were used in the experiments and showed 99% positive immunostaining with smooth muscle {alpha}-actin antibody (Sigma). The expression of AT1 receptors was confirmed by binding studies with specific receptor antagonists. For subsequent experiments, cells at 80% confluency in culture wells were used after serum depletion for 3 days.

Western blot analysis
VSMCs grown on six-well plates were stimulated with agonists for specified doses and durations. Reactions were terminated by replacement of medium with 100 µl of SDS-PAGE buffer (pH 6.8), containing 0.5 mM Tris-HCl, 2% SDS, 10% glycerol, 1% ß-mercaptoethanol, and 0.1% bromophenol blue. Following brief sonication (10 sec), lysates were boiled for 3 min at 95 C, centrifuged (14,000 x g, 5 min), and the supernatant was subjected to SDS-PAGE gel electrophoresis. Proteins in the gel were electrophoretically transferred to a nitrocellulose membrane. The membrane was then exposed to the primary antibodies overnight at 4 C. After incubation with the peroxidase linked secondary antibody for 1 h at room temperature, immunoreactive proteins were visualized by ECL reagent (19).

ERK kinase assay
VSMCs grown on 24-well plates were stimulated with agonists for 5 min. The reaction was terminated by the replacement of medium with lysis buffer (10 mM Tris-HCl, 20 mM NaCl, 2 mM EGTA, 2 mM DTT, 1 mM orthovanadate, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, pH 7.4 at 4 C). After brief sonication, samples were centrifuged at 14,000 x g for 5 min and the supernatant was assayed for ERK activity with an assay kit (BIOTRAK, Amersham Pharmacia Biotech) which measures the incorporation of [{gamma}-33P]-ATP into a specific ERK substrate peptide (KRELVERPLTPAGEAPNQALLR) as previously described (19).

Immunoprecipitation
Serum-starved VSMCs were stimulated with various agonists at 37 C. The cells were lysed with ice cold immunoprecipitation buffer (150 mM NaCl, 20 mM Tris pH 7.5, 1% Triton X-100, 5 mM EDTA, 50 mM NaF, 10% (vol/vol) glycerol and 10 µg of leupeptin, 10 µg of aprotinin, and 10 µg of phenylmethylsulfonyl, and sonicated for 5 sec. The cell lysates were centrifuged at 15,000 x g for 5 min at 4 C, and the supernatant was immunoprecipitated with antiphosphotyrosine antibody and protein A/G agarose for 3 h for the kinase assay or overnight for the immunoblotting at 4 C (25).

Immunocomplex kinase assay
The immune complexes were collected by centrifugation, washed once in immunoprecipitation lysis buffer and twice in 1x tyrosine kinase assay buffer (100 mM sodium HEPES, pH 7.6, 60 mM MgCl2, 2 mM MnCl2, 0.2 mM Na3VO4, 0.2% Triton X-100). The immune complexes were then incubated at room temperature in the kinase buffer containing 0.25 mg of the substrate poly-[Glu80-Tyr20] and 2.5 µCi of [{gamma}-32P] ATP for 15 min. The reaction mixture was spotted onto Whatman 3 mm paper, washed twice with 1% acetic acid and twice with water, and radioactivity was detected by liquid scintillation counting.

H2O2 measurement
The generation of intracellular levels of H2O2 was measured using dihydrorhodamine 123 as a probe. Briefly, serum-starved VSMCs were incubated in phenol red-free DMEM containing 10 µM dihydrorhodamine 123 for 20 min. After stimulation, cells (1.5 x 105) were detached from the culture plates by trypsin digestion, washed with PBS, and fixed in 1% paraformaldehyde. The fluorescence of rhodamine 123 in each sample was analyzed by flow cytometric analysis using an Epics Profile II flow cytometer (Coulter Electronics, Hialeah, FL) with the excitation source at 488 nm.

Statistical analysis and reproducibility of the results
Unless stated otherwise, results are representative of at least three separate experiments giving similar results. The data were analyzed by using a Student’s t test from at least three independent experiments performed in triplicate and presented as mean ± SEM. Statistical significance was shown as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of ROS inhibitors on Ang II-induced ERK activation
To demonstrate the effects of Ang II on ERK activation, we stimulated VSMCs with Ang II (100 nM) for various time periods as well as with various concentrations (1 nM-1 µM) of Ang II for 5 min, and the amount of Thr202/Tyr204-phosphorylated ERK was measured by Western blot analysis. Figure 1AGo indicates that the maximum Ang II-induced ERK phosphorylation occurs at 5–10 min. Ang II concentrations from as low as 1 nM induced ERK phosphorylation, whereas 100 nM-1 µM Ang II induced ERK phosphorylation maximally (Fig. 1BGo). 1 µM Ang II-induced ERK phosphorylation was completely inhibited by the AT1 receptor antagonist, CV11974, indicating that Ang II-induced ERK activation is mediated through the AT1 receptor in VSMCs (Fig. 1BGo).



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  		Figure 1. Effect of Ang II on ERK phosphorylation in VSMCs. A, Cells were stimulated with or without 100 nM of Ang II for the indicated time periods. Cell lysates were immunoblotted by phospho-ERK1/2 antibodies. B, Cells were stimulated with or without the indicated concentrations of Ang II for 5 min. Cell lysates were immunoblotted by phospho-ERK1/2 antibodies.

 
To investigate whether ROS are involved in Ang IIinduced ERK activation, VSMCs pretreated with or without ROS inhibitors were stimulated by Ang II (100 nM) for 5 min. As shown in Fig. 2AGo, DPI (1.25–10 µM), a frequently used inhibitor of NADH/NADPH oxidase (1, 28, 29) concentration dependently inhibited the Ang II-induced phosphorylation of ERK in VSMCs. {alpha}-Tocopherol, the major biologically active form of vitamin E, is one of the most potent natural antioxidants (30). {alpha}-Tocopherol (6.25–100 µg/ml) also inhibited the Ang II-induced ERK phosphorylation in a concentration-dependent manner (Fig. 2BGo).



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  		Figure 2. Effect of ROS inhibitors on Ang II-induced ERK activation in VSMCs. A, Cells were pretreated with or without DPI for 45 min and stimulated with 100 nM Ang II for 5 min. Cell lysates were immunoblotted by phospho-ERK1/2 and ERK2 antibodies. B, Cells were pretreated with or without {alpha}-tocopherol for 60 min and stimulated with 100 nM Ang II for 5 min. Cell lysates were immunoblotted by phospho-ERK1/2 and ERK2 antibodies. C, Cells were pretreated with or without 100 µg/ml {alpha}-tocopherol for 60 min or 10 µM DPI for 45 min, stimulated with 100 nM Ang II for 5 min, and ERK activity of cell lysates was measured by in vitro kinase assay. D, Cells were pretreated with or without 5 µM of DPI for 45 min, stimulated with 100 nM Ang II for 2.5 min, and H2O2 production was determined. Data are expressed as fold basal in which basal is defined as 1.0 in nonstimulated cells. An asterisk denotes ERK activity or H2O2 production significantly less than nonpretreated stimulated control (*, P < 0.05).

 
To further assess the involvement of ROS in Ang IImediated ERK signaling, we next examined the effect of DPI and {alpha}-tocopherol on Ang II-induced ERK enzyme activity in VSMCs. As shown in Fig. 2CGo, DPI (10 µM) and {alpha}-tocopherol (100 µg/ml) markedly inhibited Ang II-induced ERK activation in VSMCs. Moreover, pretreatment of DPI (5 µM) was sufficient to attenuate H2O2 production induced by Ang II in VSMCs (Fig. 2DGo). These data suggest a critical role for ROS in Ang II-induced ERK activation in VSMCs.

Effect of ROS inhibitors on EGF-induced ERK activation
We have previously shown that Ang II-induced ERK activation requires PTK activation in VSMCs (19). To investigate the involvement of ROS in this Ang II-induced signaling cascade, we tested the effects of ROS inhibitors on ERK activation induced by the receptor tyrosine kinase agonist, EGF in VSMCs. Both DPI and {alpha}-tocopherol failed to inhibit EGF-induced ERK activation as assessed by phospho-ERK antibody (Fig. 3AGo) and a kinase assay (Fig. 3BGo). These data suggest that ROS may act upstream of PTK activation by Ang II in VSMCs.



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  		Figure 3. Effect of ROS inhibitors on EGF-induced ERK activation in VSMCs. A, Cells were pretreated with or without {alpha}-tocopherol for 60 min or DPI for 45 min, and stimulated with 100 ng/ml EGF for 5 min. Cell lysates were immunoblotted by phospho-ERK1/2 and ERK2 antibodies. B, Cells were pretreated with or without 100 µg/ml {alpha}-tocopherol for 60 min or 10 µM DPI for 45 min, and stimulated with 100 ng/ml EGF for 5 min.

 
Effect of ROS inhibitors on Ang II-induced protein tyrosine phosphorylation and PTK activation
To investigate the possible link between ROS and a PTK in VSMCs, we studied the effect of ROS inhibitors on Ang II-induced protein tyrosine phosphorylation and PTK activation. Ang II rapidly induced tyrosine phosphorylation of several proteins in which two major bands appeared at 120 kDa (p120) and 70 kDa (p70). Ang II-induced p120 and p70 phosphorylations were partially, but significantly, inhibited by both DPI and {alpha}-tocopherol (Fig. 4Go, A and B). As shown in Fig. 5AGo, Ang II rapidly increased PTK activity (maximally 2 min) detected in immunocomplex with antiphosphotyrosine antibody. Figure 5BGo reveals that these immune complex precipitants contain p120 and p70 that are stimulated by Ang II between 2 and 5 min. Moreover, DPI (10 µM) almost completely inhibited the Ang II-induced PTK activity in VSMCs (Fig. 5CGo). These data indicate the possible involvement of a ROS-sensitive PTK in the Ang II-induced signaling cascade in VSMCs.



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  		Figure 4. Effect of ROS inhibitors on Ang II-induced PTK phosphorylation in VSMCs. A, Cells were pretreated with or without 10 µM DPI for 45 min or 50 µg/ml {alpha}-tocopherol for 60 min, and stimulated with 100 nM Ang II for the indicated time periods. Cell lysates were immunoblotted by antiphosphotyrosine antibody to detect tyrosine-phosphorylated proteins. Arrows denote 120 kDa and 70 kDa tyrosine-phosphorylated proteins. B, Gel bands in (A) were quantified using an Alpha Imager 2000 (Alpha Innotech Corp.). Graphs depict relative fold phosphorylation of p120 and p70 compared with control. An asterisk denotes phosphorylation significantly less than stimulated nonpretreated control (*, P < 0.05).

 


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  		Figure 5. Ang II-induced PTK activation in VSMCs. A, Cells were stimulated with 100 nM Ang II for the indicated time periods, and PTK activity detected in immunocomplex with antiphosphotyrosine antibody was determined. B, Cells were stimulated by 100 nM Ang II for the indicated time periods. Cell lysates were either immunoprecipitated with antiphosphotyrosine antibody or not, and immunoblotted by antiphosphotyrosine antibody. Arrows denote 120 kDa and 70 kDa tyrosine-phosphorylated proteins. C, Cells were pretreated with or without 10 µM DPI for 45 min and stimulated with 100 nM Ang II for 2 min, and PTK activity was determined. An asterisk denotes PTK activity significantly greater than nonstimulated control (A) or PTK activity significantly less than nonpretreated stimulated control (C) (*, P < 0.05).

 
H2O2-induced activation of PTK and ERK in VSMCs
To further ascertain by an alternative approach that ROS are involved in PTK and ERK activation by Ang II in VSMCs, we stimulated VSMCs with H2O2 or the thiol-oxidizing agent, diamide, and then measured protein tyrosine phosphorylation and ERK phosphorylation. As shown in Fig. 6Go, A and B, H2O2 (200 µM) and diamide (1 mM) rapidly (within 5 min) and significantly increased protein tyrosine phosphorylation of p120 and p70 maximally at 20 min approximately 4- to 5-fold and 1.5- to 2.5-fold, respectively. Treatment of VSMCs with H2O2 for 5 min significantly elevated PTK activity detected in immunocomplex with antiphosphotyrosine antibody (Fig. 7Go).



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  		Figure 6. Effect of H2O2 and diamide on PTK phosphorylation in VSMCs. A, Cells were treated with 200 µM H2O2 or 1 mM diamide for the indicated time periods. Cell lysates were immunoblotted with antiphosphotyrosine antibody. B, Gel bands in panel A were quantified using an Alpha Imager 2000 (Alpha Innotech Corp.). Graphs depict relative fold phosphorylation of p120 and p70 compared with nonstimulated control. An asterisk denotes phosphorylation significantly greater than nonstimulated control (*, P < 0.05).

 


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  		Figure 7. Effect of H2O2 on PTK activity in VSMCs. Cells were stimulated with 200 µM H2O2 for 5 min and PTK activity was measured by the immunocomplex kinase assay. An asterisk denotes PTK activity significantly greater than nonstimulated control (*, P < 0.05).

 
H2O2 and diamide also induced ERK phosphorylation as rapidly as 5 min and maximally at 10–20 min (Fig. 8Go). These results strongly support the possibility that ROS represent second messengers induced by Ang II that are capable of activating PTK and ERK in VSMCs.



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  		Figure 8. Effect of H2O2 and diamide on ERK phosphorylation in VSMCs. Cells were treated with 200 µM H2O2 or 1 mM diamide for the indicated time periods. Cell lysates were immunoblotted with phospho-ERK1/2 and ERK2 antibodies.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major findings of the present study are: 1) ROS play a critical role in the Ang II-induced ERK activation in VSMCs; and 2) the primary target of ROS produced by Ang II may be the PTK activity associated with p120 and/or p70. To our knowledge, this is the first publication linking Ang IIinduced PTK activity with ROS. In this study, the NADH/NADPH oxidase inhibitor, DPI, inhibited not only ROS production, but also the ERK activation by Ang II. The ROS scavenger, {alpha}-tocopherol, also inhibited ERK1/2 activation by Ang II in VSMCs. These data suggest that ROS produced by NADH/NADPH oxidase contribute to the Ang II-induced ERK activation in VSMCs. In support of this notion, Griendling et al. (11) showed that inhibition of NADH/NADPH oxidase by DPI could effectively abolish Ang II-induced ROS production as well as hypertrophy in VSMCs.

Earlier studies showed that H2O2 stimulated proto-oncogene expression and DNA synthesis in VSMCs (31). Consistent with the report that H2O2 stimulates ERK in VSMCs (32), we have further demonstrated that exogenous H2O2 or the thiol-oxidizing agent, diamide, can induce ERK activation in our VSMCs. Although, H2O2 has been shown to activate other members of the MAPK family, it is ERK that is believed to represent a major redox-sensitive MAPK in response to H2O2 as evident in NIH 3T3 cells (32). Moreover, H2O2 concentration and time dependently stimulated MEK1/2, the upstream activator of ERK in HeLa cells (33).

Therefore, it is reasonable to speculate that H2O2 generated through NADH/NADPH oxidase-dependent O2.- production mainly participates in Ang II-induced ERK activation in VSMCs.

Data shown in Fig. 2DGo and Fig. 1AGo indicate the presence of both ROS-dependent and independent mechanisms for ERK activation by Ang II: 1) 5 µM DPI completely inhibited ROS production; and 2) Ang II-induced ERK activation occurs more rapidly than H2O2 and diamide. Taken together, the difference in time dependency may indicate that ROS-independent ERK activation by Ang II precedes the ROS-dependent activation by Ang II. However, different experimental conditions used in these studies should also be considered.

We and others have shown that PTKs play a critical role in Ang II-induced ERK activation in VSMCs: these include c-Src, PYK2/CAKß, and EGF receptor (19, 20, 25, 26). In this study, we have demonstrated that rapid protein tyrosine phosphorylation induced by Ang II is inhibited by ROS inhibitors and that the actions of H2O2 mimics the tyrosine phosphorylation in VSMCs. We also showed that H2O2 was able to enhance PTK activity and that DPI inhibited Ang II-induced PTK activation in VSMCs. Based on these results, we propose that there exists a strong link between ROS and PTK that may positively regulate ERK activation. Recently, Src family tyrosine kinase and JAK2 were shown to mediate ERK activation by H2O2 in mouse embryo fibroblasts (34) and neonatal rat cardiac myocytes (35). In VSMCs, H2O2 was shown to activate the EGF receptor (36). Although, the mol wt of p120 and p70 are not totally matched to the PTKs previously known to be activated by Ang II or H2O2, these proteins could represent ROS-sensitive PTKs that thereby activate ERK in VSMCs.

In the present study, DPI completely inhibited PTK activation by Ang II, whereas inhibition of p120 and p70 phosphorylation by DPI was partial. A time difference exists between the p120 and p70 phosphorylation and PTK activation by Ang II. Also, the time course of p120 and p70 phosphorylation and the migration patterns of protein in the total lysate and immunoprecipitate are not exactly matched. No further data are available to reveal the exact mechanism of these discrepancies; however, the following mechanisms can be applied: 1) Similar to the ERK activation, both ROS-dependent and independent mechanisms mediate Ang II-induced tyrosine phosphorylation of p120 and p70; and 2) p120 and p70 contain several proteins including PTKs, and some of these proteins are not immunoprecipitable by the antiphosphotyrosine antibody.

Although the precise mechanisms by which ROS activate PTK are not yet clear, protein tyrosine phosphatases containing a cysteine residue in their activation site may be a direct target of ROS, which in turn, activate PTKs. Knebel et al. (37) showed that H2O2 and various other thiol-oxidizing agents could inhibit dephosphorylation of the EGFR through protein tyrosine phosphatase inhibition. Thus, the possible involvement of protein tyrosine phosphatase in the ROS-sensitive ERK activation warrants further investigation.

Contrary to our data, Baas and Berk (38) showed that only O2.- and not H2O2 was capable of activating ERK1/2 in VSMCs. Ushio-Fukai et al. (29) also showed that DPI inhibited p38 MAPK, but not ERK stimulated by Ang II in VSMCs. Although no data were available to explain these discrepancies, they may be due to the different experimental methods and/or phenotypes of VSMCs. Further studies are needed that will enable us to understand the role of ROS in the overall growth promotion by Ang II in VSMCs.

In summary, we have shown that antioxidants inhibit both PTK and subsequent ERK activation by Ang II, and that ROS can induce PTK and ERK activation in VSMCs. These data indicate that ROS play a critical role in the Ang II-induced growth-promoting signal in VSMCs, and provides significant insight into the molecular mechanisms underlying vascular remodeling.


   Acknowledgments
 
We thank Kunie Eguchi, Trinita Fitzgerald, and Sherrell Stokes for their excellent technical assistance.


   Footnotes
 
1 This research was supported in part by several National Institutes of Health Grants: HL-03320, HL-07864, HL-58205, and P-200-A-970110. Back

Received January 13, 2000.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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