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2004 CARDIOVASCULAR AND KIDNEY INVESTIGATORS MEETING

Angiotensin II-induced hypertrophy is potentiated in mice overexpressing p22^phox in vascular smooth muscle

Division of Cardiology, Department of Medicine, Emory University, Atlanta, Georgia

Submitted 26 June 2004 ; accepted in final form 25 August 2004

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Increased reactive oxygen species (ROS) are implicated in several vascular pathologies associated with vascular smooth muscle hypertrophy. In the current studies, we utilized transgenic (Tg) mice (Tg^p22smc) that overexpress the p22^phox subunit of NAD(P)H oxidase selectively in smooth muscle. These mice have a twofold increase in aortic p22^phox expression and H₂O₂ production and thus provide an excellent in vivo model in which to assess the effects of increased ROS generation on vascular smooth muscle cell (VSMC) function. We tested the hypothesis that overexpression of VSMC p22^phox potentiates angiotensin II (ANG II)-induced vascular hypertrophy. Male Tg^p22smc mice and negative littermate controls were infused with either ANG II or saline for 13 days. Baseline blood pressure was not different between control and Tg^p22smc mice. ANG II significantly increased blood pressure in both groups, with this increase being slightly exacerbated in the Tg^p22smc mice. Baseline aortic wall thickness and cross-sectional wall area were not different between control and Tg^p22smc mice. Importantly, the ANG II-induced increase in both parameters was significantly greater in the Tg^p22smc mice compared with control mice. To confirm that this potentiation of vascular hypertrophy was due to increased ROS levels, additional groups of mice were coinfused with ebselen. This treatment prevented the exacerbation of hypertrophy in Tg^p22smc mice receiving ANG II. These data suggest that although increased availability of NAD(P)H oxidase-derived ROS is not a sufficient stimulus for hypertrophy, it does potentiate ANG II-induced vascular hypertrophy, making ROS an excellent target for intervention aimed at reducing medial thickening in vivo.

reactive oxygen species; oxidant signaling; hypertrophy; vascular smooth muscle cells

Angiotensin II (ANG II) is a well-established and potent mediator of disease-related vascular wall remodeling, an integral part of which is VSMC hypertrophy (17). ANG II regulates the activity of VSMC NAD(P)H oxidases on at least two levels. Not only are NAD(P)H oxidases activated acutely via phosphorylation cascades (19), but also long-term treatment of rodents with ANG II leads to an upregulation of p22^phox and its catalytic subunits Nox1 and Nox4 (2, 14). Wang et al. (22) have shown that ANG II-induced hypertrophy is decreased in gp91^phox–/– mice, implicating NAD(P)H oxidases in the hypertrophic response to ANG II. However, the role of media-derived ROS remains unclear.

Because oxidant stress is so closely related to vascular pathology, it has been difficult to study the effect of chronic elevation in ROS in the absence of other disease-promoting stimuli. Excessive ROS may be sufficient to initiate disease progression or they may be harmful only in the context of other pathogenic events. In the current study, we used mice that overexpress p22^phox exclusively in smooth muscle [Tg^p22smc (11)] to investigate the role of smooth muscle oxidases in vascular hypertrophy. We found that increasing ROS production in the media has little effect by itself but clearly potentiates the hypertrophic response to ANG II. These findings indicate that elevated NAD(P)H oxidase expression in pathologies such as hypertension or atherosclerosis may contribute significantly to abnormal vascular growth and remodeling.

	MATERIALS AND METHODS

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Animals. Mice used in the current study overexpress p22^phox specifically in their smooth muscle (11). To generate these animals, p22^phox was cloned downstream of the smooth muscle -actin promoter fragment SMP-8 and upstream of a SV40 poly A fragment. The construct was linearized and injected into eggs, and mice were screened for insertion of p22^phox. Founder mice were then backcrossed 10 generations to the C57BL/6J strain. For the current experiments, mice that were heterozygous for p22^phox overexpression in smooth muscle (Tg^p22smc) and their negative littermate controls were used. Genotyping was determined using polymerase chain reaction by amplifying the SV40 promoter (a unique sequence found only following insertion of the transgene). Studies were completed in adult male mice, ages 12–18 wk. All animal experiments were approved by the Emory University Animal Care and Use Committee.

Infusion protocols. Mice were anesthetized, and osmotic pumps containing 100 µl of either saline or ANG II (Sigma; St. Louis, MO) were implanted subcutaneously. ANG II was administered at a dose of 0.7 mg·kg^–1·day^–1. During infusion, blood pressure was determined using standard tail-cuff methods (Visitech; Apex, NC). Additional groups of control and Tg^p22smc mice were coinfused with ebselen (Cayman Chemical; Ann Arbor, MI) dissolved in 50% DMSO administered at a dose of 10 mg·kg^–1·day^–1 via a second osmotic pump. Control mice received 50% DMSO alone. It should be noted that in vivo, ebselen exists mainly as the thiol-bound form, of which the primary cellular action is as a glutathione peroxidase mimetic. However, ebselen can also inhibit other sources of ROS, such as lipoxygenases, protein kinase C, and NAD(P)H oxidases (18).

Western blotting analysis and immunofluorescence. For both procedures, animals were euthanized by CO₂ inhalation after the infusion protocol was completed. Aortas were carefully excised, and adventitia was removed. For Western blot analysis, tissues were minced, proteins were extracted, and Western blot analysis was completed as described previously (16). Membranes were incubated with a mouse monoclonal antibody that recognizes p22^phox or a rabbit polyclonal antibody that recognizes Nox1 (a kind gift of Dr. H. Schmidt). The mouse monoclonal p22^phox antibody was raised against a GST-p22^phox fusion protein. It recognizes p22^phox purified from baculovirus but does not recognize GST.

Immunofluorescence was assessed using 7-µm OCT-embedded tissue sections fixed in acetone. After incubation in blocking buffer [3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS)] for 1 h, sections were incubated with rabbit polyclonal p22^phox antibody (a kind gift of Dr. Mark Quinn) (1:100 dilution in 1.0% BSA in PBS for 1 h), washed, and incubated with secondary antibody conjugated to rhodamine red for 30 min. Serial sections treated with secondary antibodies alone confirmed that fluorescence was not due to nonspecific binding. Aortas from a minimum of three animals per group were stained for p22^phox expression. Digital images of the sections were obtained using a Zeiss Axioskop.

Measurement of H₂O₂. After the infusion protocol, mice were euthanized by CO₂ inhalation. Aortas were excised, and adherent fat was removed. H₂O₂ was measured using a fluorometric horseradish peroxidase-linked assay (Amplex red assay, Molecular Probes) (24). This assay has been reported to be capable of detecting low levels of H₂O₂ production and is specific for measuring H₂O₂ generation because the fluorescence is inhibitable by catalase (12). Briefly, aortic segments (5-mm rings) were incubated with Amplex red (100 µM) and horseradish peroxidase (1 U/ml) for 30 min at 37°C in Krebs-HEPES buffer protected from light. Fluorescence was measured (excitation 530 nm, detection 590 nm), and background fluorescence, determined in a control reaction without sample, was subtracted from each value. H₂O₂ release was calculated using H₂O₂ standards and expressed as picomoles per milligram of dry tissue.

Morphometric analysis. After the animal was euthanasized, the heart and aorta were pressure perfused at 100 mmHg with 0.9% sodium chloride solution, followed by pressure fixation with a 10% formalin solution. Aortas were embedded in paraffin, cut in 5-µm cross sections, and stained with hematoxylin and eosin. Scaled digital images were obtained using a Zeiss Axioskop. To quantify wall thickness, perpendicular lines were drawn to determine the distance from internal elastic lamina to the external lamina at a minimum of eight locations of the aortic section. To determine cross-sectional wall area, the perimeters of the internal and external elastic laminas were traced. The area inside each respective perimeter was determined, and the difference between these areas was reported as the aortic wall area. All measurements were completed using National Institutes of Health Image (version 1.62), and analysis of subsequent sections from each animal validated the consistency of the quantification techniques.

Statistical analysis. Results are expressed as means ± SE. Statistical significance, assessed by ANOVA with Newman-Keuls multiple comparison posttest, was performed using GraphPad Prism software. A value of P < 0.05 was considered to be statistically significant.

	RESULTS

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Characterization of Tg^p22smc mice. To confirm the phenotype of the Tg^p22smc mice, we first verified that p22^phox is overexpressed in aortic tissue. Western blot analysis indicated that under basal conditions, p22^phox expression is increased approximately twofold compared with that in wild-type mice (Fig. 1A, top). A similar increase was observed using immunofluoresence staining (Fig. 1B, A and C), which also showed that the increase in p22^phox occurs in the media. Tg^p22smc mice have a significant increase in Nox1 expression, a crucial finding because Nox1 is the functional moiety in VSMCs that is responsible for the electron transport contributing to NAD(P)H oxidase activity (Fig 1A, bottom).

View larger version (34K): [in this window] [in a new window]   Fig. 1. p22phox expression is increased in transgenic (Tgp22smc) mice. Aortas from C57BL/6J and Tgp22smc mice were harvested following infusion of either saline or angiotensin II (ANG II) for 13 days. A: representative Western blot analysis of aortic protein extracts using a mouse monoclonal antibody that recognizes p22phox. or antibody against Nox1. B: representative aortic cross sections demonstrating the expression of p22phox by immunofluorescence following staining with a rabbit polyclonal antibody recognizing p22phox. p22phox staining is shown in C57BL6/J mice (A and B) and Tgp22smc mice (C and D) before (A and C) and after (B and D) ANG II infusion.

In accord with the increased expression of these NAD(P)H oxidase proteins, Tg^p22smc mice also demonstrate a twofold increase in basal H₂O₂ production compared with C57BL/6J mice (Fig. 2). ANG II infusion further potentiated the increase in H₂O₂ production in Tg^p22smc mice compared with its effect in wild-type mice. Infusion of the glutathione peroxidase mimetic ebselen induced a small, nonsignificant increase in H₂O₂ production in saline-infused mice but had no effect on basal H₂O₂ production in Tg^p22smc mice. As expected, ebselen prevented the exacerbated H₂O₂ production in response to ANG II in Tg^p22smc mice.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Aortic H2O2 release in C57BL/6J mice and Tgp22smc mice. Summary data for Amplex red experiments measuring the release of H2O2 in C57BL/6J mice (open bars) and Tgp22smc mice (filled bars) following infusion with saline or ANG II or coinfusion with ebselen for 13 days. Values represent means ± SE for a minimum of 5 animals per group. *Significant increase in H2O2 production vs. C57BL/6J mice (P < 0.001); significant increase in H2O2 production vs. Tgp22smc mice infused with saline (P < 0.001); significant decrease in H2O2 production vs. Tgp22smc mice receiving ANG II (P < 0.001).

p22^phox overexpression potentiates aortic hypertrophy. It has been reported in vitro that p22^phox antisense treatment inhibits ROS production in response to ANG II and that this inhibition prevents ANG II-induced hypertrophy in cultured VSMC (21). However, the phenotypic modulation that occurs in culture raises the question of whether p22^phox-containing NAD(P)H oxidases have a similar role in vivo. To test this notion, we assessed aortic hypertrophy after 13 days of ANG II infusion in Tg^p22smc mice. After saline infusion for 13 days, cross sections from C57BL/6J and Tg^p22smc mice appear similar (Fig. 3, A and C), suggesting that the basal changes in these mice do not significantly affect vascular growth. Similar to previous reports in both rats and mice (1, 22), ANG II infusion leads to significant hypertrophy of the aorta in both C57BL/6J and Tg^p22smc mice compared with mice receiving saline (Fig. 3, B and D). Interestingly, aortic hypertrophy in response to ANG II infusion was significantly increased in Tg^p22smc mice relative to C57BL/6J mice receiving ANG II, suggesting that the increased ROS generation in Tg^p22smc mice potentiated vascular growth in vivo.

View larger version (107K): [in this window] [in a new window]   Fig. 3. Aortic hypertrophy in C57BL/6J mice and Tgp22smc mice. A–D: representative hematoxylin and eosin-stained aortic cross section of C57BL/6J and Tgp22smc mouse aortas following infusion of saline (A and C) or ANG II for 13 days (B and D). ANG II infusion leads to aortic hypertrophy, which is potentiated in Tgp22smc mice relative to C57BL/6J mice.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Aortic hypertrophy in C57BL/6J mice and Tgp22smc mice. C57BL/6J (open bars) and Tgp22smc (filled bars) mice were infused with saline or ANG II or coinfused with ebselen for 13 days. After perfusion fixation, aortic wall thickness (in µm) was quantified. Values are means ± SE of a minimum 6 animals per group. *Significant increase in wall thickness vs. saline infused mice of the same genotype (P < 0.05); significant increase in wall thickness vs. C57BL/6J mice infused with ANG II only (P < 0.001); significant decrease in wall thickness vs Tgp22smc mice receiving ANG II (P < 0.001).

View larger version (13K): [in this window] [in a new window]   Fig. 5. Cross-sectional wall area in C57BL/6J mice and Tgp22smc mice. C57BL/6J (open bars) and Tgp22smc (filled bars) mice were infused with saline or ANG II or coinfused with ebselen for 13 days. After perfusion fixation, aortic cross-sectional wall ares (CSWA, in mm2) was quantified. Values are means ± SE of a minimum 6 animals per group. *Significant increase in CSWA vs. saline infused mice of the same genotype (P < 0.05); significant increase in CSWA vs. C57BL/6J mice infused with ANG II only (P < 0.001); significant decrease in CSWA vs. Tgp22smc mice receiving ANG II (P < 0.001).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this study, we demonstrate that increased oxidant stress due to overexpression of p22^phox and upregulation of Nox1 can, in conjunction with a hypertensive stimulus such as ANG II, exacerbate remodeling of large arteries. The increased NAD(P)H oxidase activity in Tg^p22smc mice has little effect on medial hypertrophy under physiological conditions, but when mice are subjected to the pathological stimulus of high ANG II concentrations, medial wall thickness and wall area are enhanced. This suggests that although oxidant stress may contribute to the pathogenesis of vascular disease, it alone is an insufficient stimulus for vascular growth.

p22^phox alone does not support superoxide production but has been clearly shown to stabilize the expression of gp91^phox(6) and Nox1 (7) in culture. Our data indicate that p22^phox overexpression in mice concomitantly upregulates Nox1, consistent with such an effect in vivo. We and others (2, 14) have previously shown that ANG II increases aortic p22^phox and Nox1 expression, similar to the results shown here (Fig. 1A). It is of interest that ANG II further increases p22^phox expression in Tg^p22smc mice, perhaps because the smooth muscle -actin promoter, from which SMP-8 is derived, is responsive to ANG II (8). Despite the large increase in p22^phox expression after ANG II, Nox1 levels were only slightly increased over basal levels in these mice, suggesting that the regulation of Nox1 by ANG II involves more that merely costabilization by p22^phox. The increase in Nox1 and p22^phox appears to result in enhanced oxidase activity, because the H₂O₂ response to ANG II is increased in Tg^p22smc mice (Fig. 2).

This increase in ROS in Tg^p22smc mice is associated with an enhanced hypertrophic response to ANG II, consistent with our previous in vitro findings (23). The ability of ebselen to block the exacerbated hypertrophy in Tg^p22smc mice strongly suggests that this effect is due to ROS. It is worth noting, however, that Tg^p22smc mice exhibit not only an increase in vascular ROS production but also an increase in eNOS expression and endothelial NO production (11). NO is known to inhibit vascular smooth muscle growth (3), suggesting that aortic hypertrophy in the Tg^p22smc mice might have been greater in the absence of this compensatory response. It is surprising that in C57BL/6J mice, ANG II did not increase H₂O₂ levels and that vascular hypertrophy is not blocked by ebselen coinfusion. There are several possible explanations for this observation. First, we only measured H₂O₂ at 13 days after ANG II infusion, and it is possible that H₂O₂ production may indeed be increased at earlier times. Arguing against this possibility is the observation that ebselen did not block hypertrophy in wild-type mice. Second, it is possible that ROS are not normally involved in ANG II-induced hypertrophy. This is unlikely, however, given the strong evidence of a requirement for NAD(P)H oxidases in ANG II-induced vascular growth both in vitro (4, 21, 23) and in vivo (22). Third, although ebselen reduces H₂O₂ levels, it does not target other ROS such as superoxide, which may also play a role in vascular growth (20). Finally, ebselen, at the concentrations used here, does not reduce H₂O₂ levels to baseline, suggesting that perhaps in wild-type mice the amount of H₂O₂ produced by ANG II is sufficient to support hypertrophy even in the presence of ebselen.

Another consideration in comparing the hypertrophic response in wild-type and Tg^p22smc mice is that the blood pressure response is higher in the transgenic mice, at least up to day 9. The increased blood pressure in the transgenic animals is presumably due to enhanced inactivation of nitric oxide by superoxide, however, so that even this potential effect of pressure on hypertrophy is ROS dependent. Indeed, analysis of pressure versus wall thickness or wall area in all animals in this study shows a strong correlation between these two factors (r = 0.78 for wall thickness and r = 0.85 for wall area). Furthermore, by day 13, the blood pressures are no longer different between the two strains, suggesting that the induced changes in wall thickness have successfully compensated for the increased pressure. Arguing against this interpretation, however, is the observation that even though ebselen was able to prevent exacerbation of ANG II-induced H₂O₂ production and hypertrophy in Tg^p22phox mice (Figs. 4 and 5), these mice still had elevated blood pressure, although this increase was less than in mice receiving ANG II alone (Table 1). Taken together, these considerations suggest that it is unlikely that either ROS or blood pressure is acting exclusively to facilitate the vascular hypertrophy in the Tg^p22smc mice.

These studies clearly demonstrate that alteration of medial redox status can influence vascular hypertrophy. Previous work has shown that production of ROS by either the endothelium or adventitia can alter medial hypertrophy (22). Each layer of the vessel wall has a different complement of NAD(P)H oxidases, which in turn is differentially regulated (9). This permits exquisitely fine control of ROS production both in terms of hormonal control and spatial responsiveness. The interplay between the gp91^phox-based NAD(P)H oxidases in the adventitia and the Nox1 or Nox4-based oxidases in the media may be of vital importance to the pathogenesis of vascular disease.

In summary, Tg^p22smc mice represent an excellent model with which to study the effect of NAD(P)H oxidase upregulation in the media. NAD(P)H oxidase expression is increased following balloon injury, in various hypertensive models, and in the shoulder of the atherosclerotic plaque (9). All of these pathologies are accompanied by multiple other triggers of vascular disease, including increases in inflammatory mediators and cytokines. In this study, we found that long-term medial oxidant stress is by itself insufficient to trigger vascular remodeling. However, in the presence of the inflammatory and hypertensive stimulus ANG II, upregulation of medial NAD(P)H oxidases plays an important role in disease initiation and progression. Our results clearly show that increased availability of NAD(P)H oxidase-derived ROS in the media potentiates ANG II-induced vascular hypertrophy, making ROS an excellent target for intervention aimed at reducing medial thickening in vivo.

	ACKNOWLEDGMENTS

 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-38206, HL-58000, and HL-60728 and an American Heart Association Scientist Development grant (to D. S. Weber).

	FOOTNOTES

 

Address for reprint requests and other correspondence: K. K. Griendling, Emory Univ., Div. of Cardiology, 319 WMB, 1639 Pierce Dr., Atlanta, GA 30322 (E-mail: kgriend{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.

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This Article Abstract Full Text (PDF) All Versions of this Article: 288/1/H37    most recent 00638.2004v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (13) Citing Articles via Google Scholar Google Scholar Articles by Weber, D. S. Articles by Griendling, K. K. Search for Related Content PubMed PubMed Citation Articles by Weber, D. S. Articles by Griendling, K. K.