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Am J Physiol Heart Circ Physiol 279: H2234-H2240, 2000;
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Vol. 279, Issue 5, H2234-H2240, November 2000

Upregulation of p67phox and gp91phox in aortas from angiotensin II-infused mice

M. Eugenia Cifuentes, Federico E. Rey, Oscar A. Carretero, and Patrick J. Pagano

Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, Michigan 48202-2689


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Although NAD(P)H oxidase-derived superoxide (O2-) is increased during the development of angiotensin II (ANG II)-dependent hypertension, vascular regulation at the protein level has not been reported. We have shown that four major components of NAD(P)H oxidase are located primarily in the vascular adventitia as a primary source of vascular O2-. Here we compare vascular levels of O2- and NAD(P)H oxidase in normotensive and ANG II-infused hypertensive mice and show that, after 7 days of ANG II infusion (750 µg · kg-1 · day-1 ip) in C57B1/6 mice, systolic blood pressure was increased compared with that after sham infusion, concomitant with increased O2- in the thoracic aorta as measured using lucigenin (25 µM)-enhanced chemiluminescence. Both p67phox and gp91phox were detectable by Western blotting in aortic homogenates, and we observed increased protein levels of NAD(P)H oxidase subunits. These ANG II-induced increases were normalized by simultaneous treatment with the AT1 receptor antagonist losartan. Moreover, the primary location of these subunits was the adventitia as detected immunohistochemically. Our results suggest that ANG II-induced increases in O2- are due to increased adventitial NAD(P)H oxidase activity, brought about by the heightened expression and interaction of its components.

hypertension; superoxide anion; free radicals; reactive oxygen species; NAD(P)H oxidase; NAD(P)H oxidoreductase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE CAPACITY OF SUPEROXIDE ANION (O2-) to impair nitric oxide (NO)-dependent vasodilatation (12, 28, 31) and enhance endothelium-dependent constriction (16, 39) has been extensively demonstrated in vitro, and several studies have shown that O2- is involved in the development of hypertension. Superoxide dismutase (SOD) was shown to lower blood pressure in spontaneously hypertensive rats (SHR) (30). In rats with acute angiotensin II (ANG II)-induced hypertension, SOD and catalase inhibited vascular hyperpermeability and cellular damage related to the hypertension (44), suggesting the involvement of O2- and hydrogen peroxide, respectively. In ANG II models of hypertension in rats and SHR, endothelium-dependent relaxations were impaired (6, 25) and endothelium-dependent contractions were enhanced (23). Moreover, treatment of SHR with angiotensin-converting enzyme inhibitors in vivo normalized aberrant ex vivo aortic relaxations and contractions (6, 25). Recent reports have shown that ANG II can stimulate O2- in phagocytes, aortic smooth muscle cells, and adventitial fibroblasts via NAD(P)H oxidases (11, 18, 34), implicating ANG II-induced O2- in these two forms of hypertension.

In the rat aorta, further studies have corroborated our finding that the adventitia is a major site of vascular O2- production and NAD(P)H oxidase component expression under basal conditions (34) and have demonstrated the involvement of adventitial O2- in the destruction of endothelium-derived NO and the development of passive tone during ANG II-dependent hypertension (42, 43). Because of the potential importance of the adventitia in the regulation of NO function and vascular physiology (46), we have focused our studies on aortic adventitial NAD(P)H oxidase in mice to study its regulation in various knockout and transgenic models. The purpose of this study was to examine the location and expression of NAD(P)H oxidase components in the mouse aorta and to investigate whether protein levels are increased by ANG II.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Lucigenin and diethyldithiocarbamate (DDC) were solubilized in physiological buffer; 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron) was solubilized in H2O. These compounds were purchased from Sigma (St. Louis, MO). Diphenylene iodonium was purchased from Biomol (Plymouth Meeting, PA) and solubilized in dimethyl sulfoxide, losartan was obtained from Merck (Whitehouse Station, NJ) and diluted in drinking water, and ANG II was obtained from Sigma and diluted in 0.9% saline with 0.01 N acetic acid. Monoclonal and polyclonal antibodies against gp91phox and p67phox (4, 7) were generously supplied by Dr. Mark Quinn (Montana State University, Bozeman, MT).

Animals and systolic blood pressure measurements. C57Bl/6 male mice, 8-9 wk old, were purchased from Taconic Farms. They were kept in the animal facility for 1 wk before surgery was performed, and blood pressure was measured daily for training and adjustment to the technique (see Surgery). Systolic blood pressure was measured on days 0 (basal), 3, 5, and 7 after surgery in awake mice with the use of a noninvasive computerized tail-cuff system (BP-2000; Visitech, Apex, NC). Mice were placed in temperature-controlled chambers (37°C), and blood pressure was recorded in 3 cycles of 10 measurements. Cycles with standard deviations >20 were discarded.

Surgery. All protocols using live animals were approved by the Henry Ford Hospital Committee for the Care and Use of Experimental Animals. Alzet mini-osmotic pumps (Alza Pharmaceutics, Palo Alto, CA) containing either vehicle (0.01 N acetic acid in saline solution) or ANG II (0.75 mg · kg-1 · day-1 for 7 days) were implanted intraperitoneally under sterile conditions. Some of the animals in the ANG II group were given losartan (30 mg · kg-1 · day-1) in drinking water. Seven days after surgery was performed, animals were killed and the thoracic and abdominal aorta were removed.

O2- production. O2- production was measured by lucigenin-enhanced chemiluminescence (ECL) assay as described previously (34, 36), except that the lucigenin concentration was decreased from 250 to 25 µM to avoid the artifactual O2- described by Liochev and Fridovich (24), a concentration that was validated by Li et al. (21). Thoracic aortic rings (0.5 cm) were cleaned and incubated for 30 min in modified Krebs-HEPES buffer of the following composition (in mM): 119 NaCl, 20 HEPES, 4.6 KCl, 1.0 MgSO4, 0.15 Na2HPO4, 0.4 KH2PO4, 5 NaHCO3, 1.2 CaCl2, and 5.5 glucose (pH 7.4) at 37°C in the presence of either 10 mM DDC alone or 10 mM DDC plus 0.1 mM diphenylene iodonium, a flavoprotein inhibitor of NAD(P)H oxidases. Rings were transferred to small plastic tubes containing 25 µM lucigenin in modified Krebs-HEPES buffer and incubated for 10 min at 37°C in the dark. After incubation, tubes were placed in a luminometer (TD-20e; Turner Designs, Sunnyvale, CA). Luminescence measurements were integrated for 30-s periods, and the cycle was repeated 9 times; the 10 values were then averaged. After 10 cycles, the cell-permeant O2- scavenger Tiron (10 mM) was added, and 15 more cycles were read; the final 8 values, which appeared to be maximally reduced, were averaged. Data were calculated as the change in the rate of luminescence per minute per milligram of tissue of values before and after Tiron and then converted to O2- (nmol · min-1 · mg tissue-1) as described previously (34).

Immunoblotting. Abdominal aortas were cleaned and homogenized in a glass-on-glass homogenizer in 10 mM Tris · HCl (pH 7.5), 100 mM NaCl, 300 mM sucrose, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 0.01 mg/ml each of leupeptin, aprotinin, and pepstatin. Fresh protease inhibitors were added at the time of homogenization. Samples were either used immediately or stored at -70°C. Homogenates were run on 10-20% gradient gels (Owl Separation Systems), transferred to nitrocellulose membranes (Amersham), and blotted with a previously characterized monoclonal antibody (1:1,000 dilution) against gp91phox (4) and monoclonal and polyclonal antibodies (1:1,000) against p67phox (7); the secondary antibody was goat anti-mouse IgG (Fab-specific, Sigma) for the monoclonal antibodies or goat anti-rabbit (Amersham) for the polyclonal antibodies. Blots were developed using ECL reagent (Amersham). Band intensity was compared by using an automated densitometer (model GS-670; Bio-Rad).

Immunohistochemistry. To prepare aortas for immunohistochemistry, we anesthetized C57Bl/6 mice, opened the thoracic cavity, and then performed perfusion transcardially by heart puncture, first with PBS and then with Formalin. Thoracic aortas were embedded in paraffin. Sections were then treated with cold acetic acid for 10 min, air-dried, washed with PBS, and blocked with 1% goat serum in PBS for 30 min at room temperature. They were incubated overnight with the same monoclonal antibodies against gp91phox and p67phox (1:500 dilution) used for Western blot analysis or with IgG 2a and IgG 1 isotype controls in PBS. After being washed with PBS, the sections were incubated in the dark for 45 min at room temperature with Fluorolink Cy3-labeled goat anti-mouse antibody (Amersham) diluted 100 times in PBS. Sections were washed with PBS, mounted, and examined by fluorescence microscopy.

Statistical analysis. Data are expressed as means ± SE, and n represents the number of animals used for each experiment performed on separate days. The significance of point differences in O2- generation was determined by a one-way ANOVA, and mean blood pressure was determined by ANOVA with repeated measures. Because of the large variability, Wilcoxon's nonparametric test was used when the effect of ANG II on p67phox and gp91phox expression was analyzed. P < 0.05 was considered statistically different.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

O2- generation in mouse thoracic aortas. ANG II infusion caused significant elevation of systolic blood pressure, and this increase was completely inhibited by the addition of losartan to the drinking water (Fig. 1). On day 7 we observed a significant increase in aortic O2- production in ANG II-treated mice that was inhibited by coadministration of losartan, an AT1 receptor antagonist (Fig. 2). Treatment of rings with diphenylene iodonium (100 µM) reduced ANG II-induced O2- to levels not statistically different from those in sham treatment (not shown, n = 5). These data were collected from rings equilibrated in the presence of 10 mM DDC to inhibit Cu,Zn SOD (36). In recent experiments with rings not treated with DDC, there was a 385 ± 89% increase in O2- levels (n = 3, P < 0.05).


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  		Fig. 1.   Systolic blood pressure was measured by tail-cuff plethysmography on day 0 (before surgery) and on days 3, 5, and 7 following implantation of mini-osmotic pumps containing vehicle (sham) or angiotensin II (ANG II); a third group was infused with ANG II while losartan was administered in the drinking water (ANG II + losartan). Data are means ± SE from 18-20 mice. *Statistical significance vs. sham by ANOVA; Holm's method was used to adjust the significance level for multiple comparisons.



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  		Fig. 2.   O2- production by mouse aortic rings. After 7 days of treatment with vehicle (sham), ANG II, or ANG II + losartan, ex vivo aortas were subjected to lucigenin-enhanced chemiluminescence as described in METHODS. Data are means ± SE derived from aortas of 6-7 mice. *Statistical significance vs. sham; **statistical significance vs. ANG II by one-way ANOVA; Holm's method was used to adjust the significance level for multiple comparisons.

Western blot expression of gp91phox and p67phox. We tested whether the infusion of ANG II would increase expression of NAD(P)H oxidase components in mouse aortas. Western blots of aortic homogenates from vehicle-infused mice treated with monoclonal antibody against human gp91phox exhibited a cross-reactive band of ~77 kDa (Fig. 3A), a value less than that reported for human gp91phox but higher than that expressed from the mouse phagocyte gp91phox clone (2); these discrepancies are likely related to varying degrees of glycosylation (2). The intensity of this band was increased in homogenates from ANG II-infused mice, whereas with ANG II plus losartan-treated mice, the intensity was not different from that in sham treatment (Fig. 3, A and B, n = 11). Membranes exposed to the monoclonal or polyclonal antibody to p67phox demonstrated cross-reactivity at ~67 kDa, a value that was enhanced in homogenates from ANG II-treated mice and not different from sham-treated mice given ANG II plus losartan (Fig. 4).


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  		Fig. 3.   Western blot of mouse aortic gp91phox. Mice were treated with vehicle (sham), ANG II, or ANG II + losartan. Aortic homogenates were subjected to SDS gel electrophoresis, transferred to nitrocellulose, and blotted with a monoclonal antibody to gp91phox. A: a representative Western blot of aortic homogenates from the 3 treatment groups showing gp91phox homolog cross-reactivity at ~77 kDa. B: cumulative densitometric comparisons (n = 10). Density units were collected from immunoblots; sham values in each experiment were designated as 100%. *Statistical significance determined by nonparametric Wilcoxon test comparing ANG II and sham (P < 0.01).



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  		Fig. 4.   Western blot of mouse aortic p67phox. Membranes were prepared as described in Fig. 3 and were blotted with a monoclonal antibody to p67phox. Density units were collected from immunoblots. Sham values in each experiment were designated as 100%. Other group averages were compared with the sham average and expressed as a percentage of sham control (mean ± SE) for each group (n = 11). *Statistical significance determined by Wilcoxon t-test comparing ANG II and sham (P < 0.05).

Localization of NAD(P)H oxidase in control mouse aortas. We have previously documented NAD(P)H oxidase in the adventitia of both rabbit and rat blood vessels (34, 43). Using the same antibodies and similar techniques, we localized two components of NAD(P)H oxidase, p67phox and gp91phox, to the aortic adventitia of C57Bl/6 mice. Figure 5 shows fixed cross sections of mouse aorta exposed to monoclonal antibodies against p67phox (Fig. 5A) and gp91phox (Fig. 5B), followed by incubation with a fluorescence-tagged secondary antibody. Control isotype IgG did not show cross-reactivity (not shown).


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  		Fig. 5.   Location of NAD(P)H oxidase proteins in the mouse aorta. Aortic sections were incubated with monoclonal antibodies against p67phox (A) or gp91phox (B) followed by fluorescence-tagged secondary antibody. Original magnification, ×400. The sections shown are representative of 3 experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

These are the first studies to our knowledge showing that ANG II increases aortic protein levels of NAD(P)H oxidase components concomitant with increased O2- production and elevations in blood pressure, and they strongly suggest that NAD(P)H oxidase can be regulated at the protein level. These data support previous findings in rats and rabbits showing that ANG II stimulates vascular tissue to produce NAD(P)H oxidase-derived O2- (11, 42) while corroborating our previous findings that the adventitia is the major site of vascular NAD(P)H oxidase (34, 42, 43).

A variety of studies have documented induction of vascular O2- production by ANG II and characterized the source as NAD(P)H oxidase (11, 14, 34, 38). The data reported here extend those findings to the mouse aorta. Previous measurements of O2- and induction by ANG II using lucigenin in the range of 250-500 µM have been questioned because artifactual production of O2- by lucigenin was observed under conditions that skew quantification of enzyme-derived O2- (24). Recent reports have demonstrated that concentrations in the range of 5-50 µM do not exhibit such artifactual O2- generation (21), and thus we employed 25 µM lucigenin for all measurements. Consistent with previous findings in rats and rabbits (11, 34, 42), ANG II increased O2- production that was blocked by diphenylene iodonium, the flavoprotein inhibitor of NAD(P)H oxidases, indicating increased oxidase activity. The fact that the AT1 receptor antagonist losartan was able to block this increase confirms previous reports in rats that the AT1 receptor mediates the aortic response to ANG II (38). Most studies were carried out in aortic rings with SOD inhibited; however, we did detect increases in basal O2- in these aortas. There was a greater degree of variability in basal O2- that may be explained by differing induction of SOD, which could compensate for the increased production of O2-. In fact, Fukai et al. (9) recently reported that similar doses of ANG II increase extracellular Cu,Zn SOD activity in this strain of mice at 7 days of treatment.

Phagocyte NAD(P)H oxidase is a multicomponent enzyme complex that includes the two membrane-spanning polypeptide subunits, p22phox and gp91phox (which together comprise flavocytochrome b558), and three cytoplasmic polypeptide subunits, p40phox, p47phox, and p67phox (8, 45). Moreover, the cytosolic guanine nucleotide-binding protein Rac2, a member of the Ras family of peptides, is required for oxidase activation (17). Exposure of the cell to a variety of agonists stimulates the association of cytosolic and membrane-associated components and causes activation of the normally dormant and unassembled oxidase (8, 45). It is generally well accepted that dormant phagocyte NAD(P)H oxidase is activated by the assembly of constitutively expressed components. We believe that vascular NAD(P)H oxidase may differ in at least two ways: 1) the enzyme is constitutively assembled and active under basal conditions; and 2) the components can be upregulated in response to hormonal stimulation, leading to further assembly and enhanced oxidase activity. The first contention is supported by our findings that nonstimulated aortas containing the four major subunits of NAD(P)H oxidase possess NAD(P)H oxidase activity and that immunodepletion of p67phox from aortic membranes inhibits this activity (34, 36). Second, we and other groups have shown increased mRNA for p67phox and p22phox and inferred that these increases resulted in heightened O2--generating activity (10, 33). However, the presence of mRNA for a particular protein does not necessarily mean that the corresponding protein itself is present (37). In fact, without detection of NAD(P)H oxidase protein expression (34), involvement of NAD(P)H oxidase in vascular O2- production is less clear. In the current study, Western blots showed a significant enhancement of both p67phox and gp91phox homologs in aortic homogenates from ANG II-infused versus vehicle-infused and ANG II plus losartan-treated mice that correlated positively with changes in O2- production, supporting the suggestion of transcriptional and/or translational regulation of these components. These increases would be expected to contribute to higher NAD(P)H oxidase O2--generating activity, because increases in individual components have been shown to enhance NAD(P)H oxidase activity in cell-free assays (8).

Previous reports by other groups have shown that NAD(P)H oxidase(s) exist in vascular smooth muscle cells (11, 27) and endothelial cells (15, 26). An NADH oxidase found in vascular smooth muscle cells of the rat aorta seems to interfere with vascular relaxation (38), and these cells express the mRNA for one of the cytochrome b558 subunits found in phagocyte membranes, p22phox (40). In cultured rat aortic smooth muscle cells, NAD(P)H oxidase O2- activity is stimulated by ANG II and appears to be involved in both the hypertrophic response (11, 40) and the development of hypertension (20, 38). However, most studies have focused on particular vascular segments or isolated cell types in culture and did not determine the in situ location of this activity under basal and stimulatory conditions. We have taken a broader approach using nitroblue tetrazolium staining and immunohistochemistry and have shown that the adventitia is the major site of O2- production and cross-reactivity for all four major components of NAD(P)H oxidase in the rabbit aorta (34, 36). Using in situ hybridization with cDNA for p22phox, Fukui et al. (10) showed that its mRNA was located in the media and adventitia, yet they did not detect protein cross-reactivity of rat aortic tissue or smooth muscle with antibodies raised against NAD(P)H oxidase components. Our group also showed (using two distinct O2--detection assays) that, in the rat, the adventitia is a major site of O2- production and NAD(P)H oxidase component protein expression under basal conditions and ANG II stimulation. Moreover, those studies implicated adventitial O2- in the destruction of endothelium-derived NO and the development of passive tone during ANG II-dependent hypertension (42, 43). In the present studies, we have confirmed those findings in the mouse aorta by showing localization of gp91phox and p67phox expression in the adventitia.

A large adventitial O2- source is highly relevant to the bioactivity of endothelium-derived NO. Beckman and Koppenol (1) described O2- as one of three major scavengers of NO that act as a sink and lower its bioactive concentrations over its diffusion radius of 100-200 µM (19). This phenomenon is related to the ability of NO to diffuse faster than it reacts with most biological substances. Thus endothelial NO can rapidly diffuse to the adventitia and be inactivated by O2- (because the adventitia exists within the diffusion radius of NO). Because of its relatively high O2--generating capacity compared with other vascular sources, the adventitia could be expected to be a major impediment to NO bioactivity throughout the vessel wall.

The adventitia has primarily been thought of as providing structure and neural innervation to the underlying medial smooth muscle (13, 46), while its potential importance in the regulation of NO-dependent vasodilatation and growth has long been ignored. Much of the recent interest in the adventitia has been related to the therapeutic success of endothelial NOS gene transfer in restoring impaired vascular function (5, 32). However, our studies have demonstrated that a major source of vascular O2- is adventitial fibroblasts, which in turn led to other studies documenting the importance of this source in the reduced bioactivity of endothelium-derived NO (42, 43) in normotensive and hypertensive rats. Together these findings imply a physiological role for the adventitia in regulation of vascular tone. O2- from this source may also affect vascular NO derived from medial smooth muscle cells (3, 29, 41) or from nitrergic neurotransmission, which mediates arterial relaxation (22). A resultant increase in peroxynitrite from rises in NO and O2- may increase vascular tone in some vascular beds but not in others, perhaps related to tissue-dependent differences in guanylate cyclase activation (35). Studies are ongoing to specifically address the involvement of adventitial NAD(P)H oxidase in the development of vascular tone.


    ACKNOWLEDGEMENTS

We thank Drs. Marcos Alfie, Arash Kiarash, and Nagamasa Ogasawara for technical assistance with animal models and immunohistochemistry.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants R01 HL-55425 and HL-28982 and American Heart Association Grants 95011900 and 9808086W.

Address for reprint requests and other correspondence: P. J. Pagano, Division of Hypertension and Vascular Research, Rm. 7044, E & R Bldg., Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, MI 48202-2689 (E-mail: ppagano1{at}hfhs.org).

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.

Received 16 December 1999; accepted in final form 7 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 279(5):H2234-H2240
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