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Am J Physiol Heart Circ Physiol 288: H7-H12, 2005. First published October 7, 2004; doi:10.1152/ajpheart.00637.2004
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2004 CARDIOVASCULAR AND KIDNEY INVESTIGATORS MEETING

Hemodynamic and biochemical adaptations to vascular smooth muscle overexpression of p22phox in mice

Karine Laude,1 Hua Cai,1 Bruno Fink,1 Nyssa Hoch,1 David S. Weber,1 Louise McCann,1 Georg Kojda,2 Tohru Fukai,1 Harald H. H. W. Schmidt,3 Sergey Dikalov,1 Santhini Ramasamy,1 Graciela Gamez,1 Kathy K. Griendling,1 and David G. Harrison1

1Division of Cardiology, Emory University, Atlanta, Georgia; and 2Institut für Pharmakologie und Klinische Pharmakologie, Duesseldorf, and 3Rudolf-Bucheim Institut für Pharmacology, Justus-Liebig-Universtat, Giessen, Germany

Submitted 25 June 2004 ; accepted in final form 25 August 2004


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Protein levels and polymorphisms of p22phox have been suggested to modulate vascular NAD(P)H oxidase activity and vascular production of reactive oxygen species (ROS). We sought to determine whether increasing p22phox expression would alter vascular ROS production and hemodynamics by targeting p22phox expression to smooth muscle in transgenic (Tg) mice. Aortas of Tgp22smc mice had increased p22phox and Nox1 protein levels and produced more superoxide and H2O2. Surprisingly, endothelium-dependent relaxation and blood pressure in Tgp22smc mice were normal. Aortas of Tgp22smc mice produced twofold more nitric oxide (NO) at baseline and sevenfold more NO in response to calcium ionophore as detected by electron spin resonance. Western blot analysis revealed a twofold increase in endothelial NO synthase (eNOS) protein expression in Tgp22smc mice. Both eNOS expression and NO production were normalized by infusion of the glutathione peroxidase mimetic ebselen or by crossing Tgp22smc mice with mice overexpressing catalase. We have previously found that NO stimulates extracellular superoxide dismutase (ecSOD) expression in vascular smooth muscle. In keeping with this, aortic segments from Tgp22smc mice expressed twofold more ecSOD, and chronic treatment with the NOS inhibitor NG-nitro-L-arginine methyl ester normalized this, suggesting that NO regulates ecSOD protein expression in vivo. These data indicate that chronic oxidative stress caused by excessive H2O2 production evokes a compensatory response involving increased eNOS expression and NO production. NO in turn increases ecSOD protein expression and counterbalances increased ROS production leading to the maintenance of normal vascular function and hemodynamics.

transgenic animals; oxidant stress; endothelium; nitric oxide

IN MANY PATHOLOGICAL CONDITIONS associated with vascular lesion formation the production of reactive oxygen species (ROS) is increased in the vessel wall. As a consequence, nitric oxide (NO) bioavailability is decreased due in large part to its rapid reaction with superoxide (O2–·). In addition, it is now clear that ROS may have important intracellular signaling properties. As an example, H2O2 has been shown to modulate endothelial NO synthase (eNOS) protein expression in vitro (6). This upregulation of eNOS by H2O2 may occur in pathological conditions such as hypercholesterolemia and hypertension where both increased O2–· and increased eNOS expression have been observed (1, 12).

Major sources of ROS in vascular cells are the NAD(P)H oxidases, which are multisubunit enzyme complexes similar to the phagocytic oxidase. In phagocytic cells, NAD(P)H oxidases consist of the catalytic gp91phox and p22phox subunits. Recently, it has been recognized that gp91phox is a member of a larger family of Nox proteins, and it has been renamed Nox2. The vascular smooth muscle cells (VSMC) NAD(P)H oxidases differ from the phagocytic enzyme in that they utilize Nox1 and Nox4, and larger vessels do not contain Nox2 (16). However, all NAD(P)H oxidases utilize p22phox, which plays an important role as a docking protein for the cytoplasmic subunits and as a stabilizer of Nox protein expression (15). The vascular NAD(P)H oxidases can be activated by a variety of pathophysiological stimuli, including angiotensin II (ANG II) (16). In addition, over several days, ANG II increases expression of p22phox, Nox1, and Nox4. Previously, we have found that during ANG II-induced hypertension, the time course of increased NAD(P)H oxidase activity and blood pressure paralleled that of an increase in p22phox (18), suggesting that this subunit may have an important role in the modulation of overall enzyme activity. In cultured VSMC, inhibition of p22phox expression using antisense techniques inhibits hypertrophy caused by ANG II (22). Moreover, polymorphisms of p22phox have been variably associated with an increase in cardiovascular events, a reduction in endothelium-dependent vasodilatation, and an increase in coronary atherosclerosis. Intimal VSMC in atherosclerotic lesions contain large amounts of p22phox that colocalizes with sites of O2–· production (21).

Given this apparent importance of p22phox, we sought to determine whether increasing its expression in vivo would alter vascular ROS production and to understand how this would affect hemodynamics and endothelium-dependent vasodilatation by directing expression of p22phox to smooth muscle in transgenic mice. Our initial characterization of these mice showed that their vessels indeed produced excessive O2–· and H2O2, but their endothelium-dependent vasodilatation and blood pressure were normal. Additional studies indicated that expression of eNOS and extracellular superoxide dismutase (ecSOD) was increased. We propose that compensatory responses such as an increase in expression of eNOS and ecSOD are critical in the setting of oxidative stress and that overt vascular dysfunction does not occur until these fail.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
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Animals. Mice overexpressing the NAD(P)H oxidase p22phox subunit in VSMC (Tgp22smc mice) were created by cloning the p22phox cDNA downstream of the smooth muscle cell-targeted smooth muscle {alpha}-actin (SMP-8) promoter and upstream of a sarcovirus 40 (SV40) polyA fragment (Fig. 1A). The Sph1-Kpn1 fragment was used for oocyte injection. Several founder Tgp22smc mice were obtained that were able to transmit the transgene to offspring. They were backcrossed at least 10 times to the C57BL/6J background. C57BL/6J mice were used as controls. Blood pressure was measured using telemetry (Data Sciences International) as described previously (4). In some experiments, the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) was given in the drinking water (100 mg·kg–1·day–1). In other experiments, the glutathione peroxidase mimetic ebselen (10 mg·kg–1·day–1 in 50% DMSO) was infused chronically via an osmotic minipumps (Alzet; Durect, CA) implanted subcutaneously. As an alternative approach for H2O2 scavenging, we crossed Tgp22smc mice with mice overexpressing catalase driven by the Tie-2 promoter (Tgp22phox/catalase). On the day of study, the animals were killed with CO2 inhalation, and the thoracic aortas were removed and dissected free of adherent tissues. All animal experiments were approved by the Emory University Animal Care and Use Committee.



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  		Fig. 1. Creation of transgenic (Tgp22smc) mice. A: plasmid construction used for targeting of p22phox overexpression in smooth muscle cells (SMC). B: Western blot analysis of p22phox protein in aorta, heart, and lungs homogenates (left) and aortic Nox1 protein expression (right). Data are representative of 3 separate experiments.

 
Vascular reactivity. Thoracic aortas were cut into 3- to 4-mm ring segments and mounted for isometric tension recording in a water-jacketed organ bath (14). Passive tension was adjusted to 1 g, and vessels were preconstricted with 2 µM PGF2{alpha}. Relaxations to cumulative concentrations of acetylcholine were examined.

Determination of aortic O2–· and H2O2. Extracellular O2–· release was measured using the SOD-inhibitable cytochrome c reduction assay as previously described (14). To evaluate intracellular O2–·, we measured the formation of oxyethidium from dihydroethidium using HPLC as recently reported (7). H2O2 was measured using a fluorometric HRP-linked assay (Amplex red assay, Molecular Probes) as previously described (23).

Determination of aortic NO production. NO production was measured by electron-spin resonance (ESR) using the specific colloid probe Fe2+ diethyldithiocarbamate in the absence or presence of 10 µM A23187 [GenBank] (13). ESR settings were the following: microwave frequency, 9.431 GHz; modulation frequency, 100 kHz; modulation amplitude, 5 G; field sweep, 50 G; microwave power, 10 mW; conversion time, 1,312 ms; time constant, 5,248 ms; and receiver gain, 1 x 105.

Determination of aortic p22phox, Nox1, eNOS, and SOD protein levels. Western blot analyses were performed on aortic homogenates. Antibodies used were the following: a polyclonal antibody against Nox1, an eNOS monoclonal antibody (BD Transduction Laboratories), Mn-SOD and Cu/Zn-SOD polyclonal antibodies (Stressgen), a monoclonal antibody generated against the mouse p22phox protein, and a previously generated ecSOD polyclonal antibody.

Statistical analysis. All values are means ± SE. Data were compared between groups of animals by t-test when one comparison was performed and by ANOVA for multiple comparisons. When significance was indicated by ANOVA, the Tukey post hoc test was used to specify between group differences.


   RESULTS
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Characterization of the Tgp22smc mice. Compared with their C57BL/6J background, the Tgp22smc mice were overtly normal with normal litter sizes and growth rates. p22phox protein expression was increased in the aorta but not in other tissues such as the heart and lungs (Fig. 1B), confirming specific targeting of gene overexpression.

Recent studies have demonstrated that p22phox and Nox2 stabilize each other in granulocytes. We sought to determine whether overexpression of p22phox increased expression of one or more of the Nox proteins. Western blot analysis demonstrated no difference in either Nox2 (gp91phox) or Nox4 (data not shown). In contrast, expression of Nox1 was markedly increased in aortas of Tgp22smc mice (Fig. 1B).

Production of O2–· and H2O2. HPLC analysis of conversion of dihydroethidium to oxyethidium revealed that the intracellular production of O2–· was increased in aortas from Tgp22smc mice (Fig. 2A). In contrast, extracellular O2–· production detected by SOD-inhibitable cytochrome c reduction was not increased in Tgp22smc aortas (Fig. 2B). H2O2 production was significantly increased in aortas from Tgp22smc mice compared with control (Fig. 2C).



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  		Fig. 2. Aortic O2–·, H2O2, and SOD protein isoforms. A: intracellular superoxide (O2–·) measured by HPLC. Top, representative HPLC graph. Bottom, grouped data (n = 6–8 per group). au, arbitrary units. B: extracellular O2–· measured by the cytochrome c reduction assay (n = 6–8 per group). C: extracellular H2O2 (Amplex red assay, n = 6–8 per group). D: Western blots of the 3 SOD isoforms. ecSOD, extracellular SOD. Top, representative blot of 3 separate experiments. Bottom, grouped data for ecSOD. O.D., optical density. Significant differences: *P < 0.05 and **P < 0.01, t-test.

 
We also measured the expression of SOD proteins by Western blot analysis. Of the three SOD isoforms, ecSOD was increased in Tgp22smc mice by twofold (Fig. 2D).

Effect of p22phox overexpression on vascular function and hemodynamics. To determine whether the increased aortic O2–· and H2O2 affected vascular function, we measured blood pressure and aortic endothelium-dependent relaxation. Surprisingly, blood pressure was identical in Tgp22smc mice and in controls (110 ± 1 vs. 116 ± 1 mmHg in control; Fig. 3A), and endothelium-dependent vasodilatation to acetylcholine was not altered (Fig. 3B).



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  		Fig. 3. Hemodynamics and endothelial function. A: systolic arterial pressure (telemetry). B: aortic endothelium-dependent relaxation to acetylcholine (ACh) after precontraction with 2 µM PGF2{alpha} (n = 5–6 per group).

 
Effect of p22phox overexpression on NO production and eNOS protein expression. Whereas Tgp22smc mice have an increased O2–· and H2O2 production, there is no alteration in blood pressure or endothelium-dependent vasodilatation. We hypothesized that compensatory mechanisms may develop, preventing increased blood pressure and vascular dysfunction. Recent studies have shown that H2O2 potently stimulates eNOS expression and function in cultured endothelial cells (2). Because H2O2 production was increased in Tgp22smc mice, we examined eNOS expression and NO production in these animals. NO production was increased by twofold in aortas of Tgp22smc mice under basal conditions and by more than sevenfold in response to the calcium ionophore A23187 [GenBank] (10 µM, Fig. 4A). eNOS protein was twofold higher in Tgp22smc mice (Fig. 4B).



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  		Fig. 4. Aortic nitric oxide (NO) production and endothelial NO synthase (eNOS) protein expression. A: basal and stimulated NO production (electron-spin resonance, ESR). Top, representative spectra. Bottom, grouped data (n = 5–6 per group). B: Western blots of eNOS protein. Top, representative blot of 3 separate experiments. Bottom, grouped data. Significant differences: *P < 0.05, **P < 0.01, t-test.

 
To further investigate a link between H2O2 and eNOS protein upregulation, we treated mice for 7 days with ebselen to scavenge H2O2. Ebselen treatment decreased aortic H2O2 production in both control and Tgp22smc mice (Fig. 5A). Moreover, ebselen infusion decreased eNOS protein expression in both the Tgp22smc and C57BL/6J mice (Fig. 5B). Because these results obtained by a pharmacological approach suggest that H2O2 upregulates eNOS protein expression in vivo, we also examined eNOS protein in Tgp22phox/catalase mice. Expression of eNOS was normalized in these mice, clearly demonstrating that H2O2 is responsible for upregulation of eNOS protein in vivo (Fig. 5C).



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  		Fig. 5. Effect of H2O2 scavenging on eNOS protein expression. A: extracellular H2O2 after ebselen (n = 6–8 per group). B: Western blot analysis of eNOS protein after ebselen. Top, representative blot of 3 separate experiments. Bottom, grouped data. C: Western blot analysis of eNOS protein in Tgp22phox/catalase mice. Top, representative blot of 3 separate experiments. Bottom, grouped data. Significant differences: *P ≤ 0.05 vs. C57BL/6J; #P ≤ 0.05 vs. Tgp22smc.

 
Effect of NO on ecSOD protein expression. The above data indicate that overexpression of p22phox in smooth muscle increased eNOS expression, NO production, and ecSOD protein expression. Previously, we (9) have found that NO is a potent stimulus for ecSOD protein expression in vitro. We next determined whether increased NO production in Tgp22smc mice underlies the increased ecSOD expression. L-NAME treatment (100 mg·kg–1·day–1) for 14 days significantly increased blood pressure in C57BL/6J and Tgp22smc mice (Fig. 6A) and abolished basal and stimulated NO production (Fig. 6B). L-NAME also significantly decreased ecSOD protein expression in Tgp22smc mice but not in C57BL/6J mice (Fig. 6C).



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  		Fig. 6. Effect of NO inhibition on extracellular SOD (ecSOD) protein expression. A: mean arterial pressure (MAP) monitored by telemetry (n = 5–6 per group). B: basal and stimulated NO production (ESR, n = 6–8 per group). L-NAME, NG-nitro-L-arginine methyl ester. C: Western blot analysis of ecSOD protein after L-NAME. Top, representative blot of 3 separate experiments. Bottom, grouped data. Significant differences: *P < 0.05 and **P < 0.01 vs. untreated; {dagger}P < 0.05 and {dagger}{dagger}P < 0.01 vs. C57BL/6J.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we sought to determine whether increasing smooth muscle expression of p22phox would enhance vascular ROS production and to examine the effect this has on endothelium-dependent vasodilatation and hemodynamics. We found that aortas of Tgp22smc mice produce increased O2–· and H2O2 but have no change in endothelium-dependent vasodilatation or hemodynamics. Tgp22/smc mice also had a striking increase in vascular NO production and eNOS expression and an increase in ecSOD expression. From studies using L-NAME, an inhibitor of NO production, ebselen, a glutathione peroxidase mimetic to scavenge H2O2, and mice overexpressing both p22phox and catalase, we propose that chronic overproduction of O2–· and H2O2 leads to a compensatory pathway similar to that illustrated in Fig. 7.



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  		Fig. 7. Proposed compensatory pathway in response to chronic vascular oxidative stress.

 
One interesting finding in this study is that overexpression of p22phox was associated with an increase in expression of the gp91phox homolog Nox1. Whereas the mechanism for this remains unclear, it is possible that increased levels of p22phox stabilize Nox1 protein. This is analogous to the situation in phagocytes where processing of Nox2 is dependent on the presence of p22phox (5). In the neutrophil, p22phox and Nox2 are not targeted to the plasma membrane individually but require one another to avoid degradation in the proteosome. Whether p22phox and Nox1 interact in this fashion in VSMC remains unclear. Nevertheless, the increase in Nox1 probably plays a critical role in the increased production of ROS in vessels of Tgp22smc mice, because this represents the catalytic subunit responsible for electron transfer to oxygen.

NAD(P)H oxidases produce O2–· based on the one-electron reduction of oxygen by its Fe2+ center, but activation of this system can also lead to the formation of H2O2, either via spontaneous dismutation or after enzymatic dismutation by one of the SOD isozymes. The mechanism responsible for the preferential production of H2O2 in vessels of Tgp22smc mice remains unclear but may be related to the increased ecSOD protein expression. Indeed, the increased intracellular O2–· production we found in vessels of Tgp22vsmc mice was not reflected in extracellular release of O2–·. Intracellularly produced O2–· can diffuse from the cell in the form of uncharged hydroperoxy radical, and we have shown that the cytochrome c assay can detect an increase in O2–· produced by uncoupled eNOS, which releases O2–· intracellularly (14). Therefore, the inability to detect increased O2–· in these experiments is unlikely due solely to the fact that O2–· was only produced intracellularly.

We found that Tgp22smc mice have a twofold increase in the vascular levels of ecSOD. This enzyme resides in the extracellular space surrounding VSMC and endothelial cells and rapidly catalyzes dismutation of O2–· to H2O2 (8). It is therefore reasonable to conclude that increased ecSOD diminish detection of O2–· from vessels of the Tgp22smc mice. We have previously shown that NO is a potent stimulus for ecSOD production in VSMC via a cGMP-dependent pathway (9). It is likely that increased NO production in the Tgp22smc mice is in part responsible for the increased ecSOD production because L-NAME treatment completely abrogated this increase while having no effects on control mice. In our previous study (9), we showed that ecSOD levels are reduced and not increased by exercise training in eNOS-deficient mice. The present study demonstrates that a chronic increase in NO can have the opposite effect, i.e., stimulating ecSOD expression.

One of the major findings of this study is that the resulting increase in H2O2 observed in Tgp22smc mice is associated with an increased eNOS protein expression and NO production. Our group (2) has shown that H2O2 upregulates eNOS mRNA and protein expression, as well as eNOS protein activity (3, 6), through a Ca2+/calmodulin kinase II and Janus kinase 2-dependent mechanism. These previous experiments were performed in cultured endothelial cells and involved administration of high concentrations of H2O2. The present study is the first to demonstrate that H2O2 can stimulate eNOS expression in vivo.

In these experiments, we used the glutathione peroxidase mimetic ebselen to reduce H2O2. Ebselen has been shown to inhibit lipoxygenases, NOSs, and protein kinase C and to prevent apoptosis (19). These effects of ebselen are inhibited by thiols and therefore less likely to account for its effects in vivo. Furthermore, our results with mice overexpressing catalase support the concept that H2O2 is likely responsible for some of the effects we observed in the Tgp22smc mice.

In additon to the twofold increase in NOS expression, we found an even greater increase in NO production by vessels of Tgp22smc mice, particularly in response to calcium stimulation. These results may help explain why Tgp22smc mice had no alteration in endothelium-dependent vasodilatation or hemodynamics despite an increase in vascular O2–· production. In addition to being a potent stimulus for NO production by endothelial cells, H2O2 can stimulate GTP-cyclohydrolase mRNA and protein expression in endothelial cells, leading to an increase in tetrahydrobiopterin levels and promoting NO production (20). The increase in ecSOD could also protect NO from degradation by O2–·, making more NO available for trapping by Fe2+ diethyldithiocarbamate. It is of interest that the p22phox transgene was targeted to smooth muscle, and thus the source of H2O2 was clearly the VSMC oxidase, likely responsible for stimulating endothelial NO production. There are many instances where paracrine factors from the endothelium, such as NO, prostacyclin, or endothelium-derived hyperpolarizing factor, regulate VSMC tone (11). In this instance, we have demonstrated a novel function of H2O2 from the VSMC exerting a paracrine effect on endothelial cells, modulating endothelial eNOS expression and NO production.

In summary, our studies in Tgp22smc mice provide insight into how vessels may respond to a prolonged increase in H2O2 production. Overexpression of the NAD(P)H oxidase p22phox subunit in VSMC is associated with increased expression of the Nox1 catalytic subunit and an increase in VSMC H2O2 production. This is associated with a series of compensatory mechanisms that preserve normal endothelial function. It is interesting to speculate that this compensatory pathway is operative in numerous conditions where oxidative stress is increased. As examples, ANG II can stimulate both increased ecSOD and NO production (3, 10), and, at least in some models of hypercholesterolemia (17), eNOS expression is increased. In many instances, these compensatory mechanisms may preserve normal vascular function, as in Tgp22smc mice. The failure of these compensatory mechanisms are therefore likely responsible for development of vascular dysfunction and initiation of disease.


   GRANTS
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grants HL-39006, HL-38206, and HL-59248; NHLBI Program Project Grants HL-58000 and HL-075209; a Department of Veterans Affairs Merit Grant, and Deutche Forshungs Komission Grant 9772179. K. Laude was supported by a grant from the French Foundation for Medical Research.


   ACKNOWLEDGMENTS
 
We express our appreciation to Dr. R. Clempus for technical help and discussion this study.

Present address of H. Cai: Section of Cardiology, Dept. of Medicine, The Univ. of Chicago, Chicago, IL 60637.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. G. Harrison, Division of Cardiology, Emory Univ., 101 Woodruff Circle, Atlanta, GA 30322 (E-mail: dharr02{at}emory.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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