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(Circulation Research. 1997;80:45-51.)
© 1997 American Heart Association, Inc.

Articles

p22phox mRNA Expression and NADPH Oxidase Activity Are Increased in Aortas From Hypertensive Rats

Toshiki Fukui, Nobukazu Ishizaka, Sanjay Rajagopalan, Jørn Bech Laursen, Quinn Capers, IV, W. Robert Taylor, David G. Harrison, Hector de Leon, Josiah N. Wilcox, Kathy K. Griendling

the Department of Medicine, Divisions of Cardiology (T.F., N.I., S.R., J.B.L., Q.C. IV, W.R.T., D.G.H., K.K.G.) and Hematology/Oncology (H. de L., J.N.W.), Emory University; The Atlanta Veteran's Affairs Medical Center (W.R.T., D.G.H.), Atlanta, Ga; and Medical Department B (J.B.L.), National University Hospital, Rigshospitalet, Copenhagen, Denmark.

Correspondence to Kathy K. Griendling, PhD, Division of Cardiology, Emory University School of Medicine, 1639 Pierce Dr, 319 Woodruff Memorial Building, Atlanta, Ga 30322. E-mail kgriend@emory.edu

	Abstract

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Recent studies suggest that superoxide production by the NADPH/NADH oxidase may be involved in smooth muscle cell growth and the pathogenesis of hypertension. We previously showed that angiotensin II (Ang II) activates a p22phox-based NADPH/NADH oxidase in cultured rat vascular smooth muscle cells and in animals made hypertensive by infusion of Ang II. To investigate the mechanism responsible for this increased oxidase activity, we examined p22phox mRNA expression in rats made hypertensive by implanting an osmotic minipump that delivered Ang II (0.7 mg/kg per day). Blood pressure began to increase 3 days after the start of Ang II infusion and remained elevated for up to 14 days. Expression of p22phox mRNA in aorta was also increased after 3 days and reached a maximum increase of 338±41% by 5 days after pump implantation compared with the value after sham operation. This increase in mRNA expression was accompanied by an increase in the content of the corresponding cytochrome (twofold) and NADPH oxidase activity (179±11% of that in sham-operated rats 5 days after pump implantation). Treatment with the antihypertensive agents losartan (25 mg/kg per day) or hydralazine (15 mg/kg per day) inhibited this upregulation of mRNA levels and activity. Furthermore, infusion of recombinant heparin-binding superoxide dismutase decreased both blood pressure and p22phox mRNA expression. In situ hybridization of aortic tissue showed that p22phox mRNA was expressed in medial smooth muscle as well as in the adventitia. These findings suggest that Ang II–induced hypertension activates the NADPH/NADH oxidase system by upregulating mRNA levels of one or several components of this oxidase system, including the p22phox, and that the NADPH/NADH oxidase system is associated with the pathology of hypertension in vivo.

Key Words: NADPH oxidase • p22phox • hypertension • angiotensin II • muscle, smooth, vascular

	Introduction

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During the past several years, it has become apparent that vascular tissues can produce superoxide anion (·O₂^-) and other oxygen-derived free radicals. The production of these reactive oxygen species is increased in pathophysiological conditions and may contribute to altered control of vasomotor tone, atherosclerosis, and hypertension.^{1 2 3}

There are several possible sources of ·O₂^- in the diseased vessel wall. Superoxide may be produced by the endothelium,⁴ smooth muscle cells,⁵ adventitial fibroblasts,⁶ or inflammatory cells that have migrated into the wall.⁷ The identity of the cellular enzymes responsible for ·O₂^- generation is also unclear; however, evidence is accumulating that the NADPH oxidase first identified in neutrophils may be important. In calf pulmonary artery, Mohazzab and Wolin⁸ showed that NADH/NADPH oxidases account for the majority of ·O₂^- generation. Furthermore, Pagano et al⁹ have recently shown that a major source of ·O₂^- in the rabbit aorta is the NADPH oxidase located in medial smooth muscle and adventitial tissues.

The molecular identity of the vascular NADPH/NADH oxidase system is only partially understood. In neutrophils, ·O₂^- production is catalyzed by the membrane-bound multicomponent NADPH oxidase system, which includes a 22-kD -subunit (p22phox) of cytochrome b₅₅₈.¹⁰ The p22phox functions as an integral subunit of the final electron transporter from NADPH to heme to molecular oxygen in generating ·O₂^-. Recently, we cloned p22phox from rat aortic VSMCs,¹¹ and using transfection of antisense p22phox, found that this subunit is a critical component of the vascular smooth muscle ·O₂^--generating NADPH/NADH oxidase.¹² Nevertheless, the nonphagocytic oxidase appears to be structurally and functionally different from the neutrophil oxidase, as manifested by the absence of the 91-kD cytochrome b₅₅₈ ß-subunit (gp91phox)¹³ and the preferential utilization of NADH rather than NADPH as the substrate in the nonphagocytic oxidase.⁵

As is the case in neutrophils, NADPH/NADH oxidase activity in vascular cells is under hormonal control. We previously demonstrated that Ang II stimulation of VSMCs results in a delayed prolonged activation of the NADPH/NADH oxidase.⁵ More recently, we have shown that the activity of this oxidase is increased in the aortas of rats made hypertensive by Ang II infusion.¹⁴ The mechanisms responsible for this increase in activity remain to be defined. In the present study, we investigated the contribution of the regulation of p22phox mRNA expression to the increased NADPH/NADH oxidase activity in hypertensive animals. We found that vascular p22phox mRNA expression and NADPH oxidase activity were elevated in rats receiving Ang II infusion and that this correlated well with blood pressure, suggesting that upregulation of p22phox levels may contribute to oxidase activation and the development of hypertension in animals with high circulating levels of Ang II.

	Materials and Methods

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Materials
Human [Val⁵]Ang II, proteinase K, RNase A, reduced Triton X-100, and hydralazine were purchased from Sigma Chemical Co. Losartan was a kind gift from Dr R.D. Smith (DuPont de Nemours Co, Wilmington, Del). Alzet osmotic minipumps were from Alza Corp. [³⁵S]UTP (specific activity, 1200 Ci/mmol) was purchased from Amersham, and [³²P]dCTP was from Du Pont NEN. NTB2 nuclear track emulsion was from International Biotechnologies, and TRI reagent was from Molecular Research Center. Rats were purchased from Harlan Sprague Dawley, Inc (Indianapolis, Ind). Magna NT nylon membranes were from Micron Separation, Inc. Prime-It II probe labeling kits were purchased from Stratagene, and Biospin P30 columns were from Bio-Rad. HB-SOD was a generous gift from Dr Bruce Freeman, University of Alabama.

Animal Models
Male Sprague-Dawley rats weighing 250 to 300 g were anesthetized with intraperitoneal ketamine (80 mg/kg) and xylazine (10 mg/kg). An incision was made in the midscapular region, and an osmotic minipump (Alzet model 2001 for 1-, 3-, and 5-day infusion or model 2002 for 8- and 14-day infusion) was implanted. Pumps contained Ang II dissolved in 0.15 mol/L NaCl containing 0.01N acetic acid, and the infusion rate was 0.7 mg/kg per day. Sham-operated rats underwent an identical surgical procedure, but either no pump or a pump containing vehicle only was implanted. In some experiments, losartan (25 mg/kg per day) or hydralazine (15 mg/kg per day) was given in the drinking water beginning 2 days before pump implantation. Systolic blood pressures were measured in conscious rats by tail-cuff plethysmography.

For experiments in which rats received a continuous infusion of HB-SOD, rats were chronically catheterized with one catheter (medical-grade Tygon catheters) implanted through the left carotid artery and advanced so that its tip was in the ascending aorta. This catheter was used to record blood pressures and, for pressure measurement, was connected to a Gould pressure transducer and an oscillographic recorder (Gould RS 3600). A second (infusion) catheter was placed in the superior vena cava via the left external jugular vein. The catheters were filled with a solution of 50% glucose and 500 IU/mL heparin and plugged with a nylon pin. Each catheter was externalized through the neck region, between the scapulae, and secured by a polyester felt disk placed subcutaneously. After catheter implantation, the rats were housed individually until they had regained their preoperative weight and appeared healthy (6 to 8 days after catheter implantation). At the end of the recovery period, rats were divided into two different treatment groups (vehicle and Ang II), and each treatment group was divided into two subgroups, one that received HB-SOD (1200 U/kg per day) and one that received vehicle alone. Three days later, rats were reanesthetized, and osmotic minipumps were implanted for infusion of Ang II or vehicle as described above.

RNA Purification From Rat Aorta
Rats were killed by lethal injection of sodium pentobarbital. The aorta was quickly excised, the periadventitial tissues were carefully removed, and the vessels were immediately frozen in liquid nitrogen. The frozen aorta was homogenized in TRI reagent, and chloroform was added to the lysates. After centrifugation, the upper layer was taken and subjected to isopropanol precipitation. Final total RNA concentration and purity was assessed by measurement of absorbances at 260 and 280 nm. Samples were stored at -80°C until use.

Northern Blot Hybridization
Electrophoresis was performed on 1% denaturing formaldehyde agarose gels. Total RNA (20 µg) was loaded in each lane. After electrophoresis, RNA was transferred to Magna NT nylon membranes. After immobilization by UV cross-linking, blots were prehybridized for at least 4 hours at 42°C in the following solution: 1 mol/L NaCl, 50 mmol/L Tris, 5x Denhardt's solution, 50% formamide, 0.5% SDS, and 100 µg/mL sheared and denatured salmon sperm DNA. Probe labeling was performed by the random primer–labeling method with [³²P]dCTP using the Prime-It II kit. Unincorporated ³²P was removed using Biospin P30 columns. The probe used was a full-length cDNA for rat p22phox, as described previously.¹¹ After autoradiography, the relative density of each band was determined using laser densitometry. The density of individual bands was normalized to 28S ribosomal RNA.

NADPH Oxidase Assay in Aorta
NADPH oxidase activity in aortic tissue was measured 5 days after implantation of Ang II–containing pumps. Some animals also received losartan (25 mg/kg per day) or hydralazine (15 mg/kg per day) treatment, as described above. Aortic segments (2 to 3 cm) were placed in a chilled modified Krebs/HEPES buffer containing (mmol/L) NaCl 99.01, KCl 4.69, CaCl₂ 1.87, MgSO₄ 1.20, K₂HPO₄ 1.03, NaHCO₃ 25.0, sodium HEPES 20.0 and glucose 11.1, pH 7.4. Periadventitial tissue was carefully removed, and the vessels were repeatedly washed to remove adherent blood cells. A 10% vessel homogenate was prepared in a 50 mmol/L phosphate buffer containing 0.01 mmol/L EDTA by homogenizing in a glass-to-glass motorized homogenizer. The homogenate was then subjected to low-speed centrifugation (1000g) for 10 minutes to remove unbroken cells and debris. Supernatants (20 µL) were added to glass scintillation vials containing 250 µmol/L lucigenin in 2 mL phosphate buffer. The chemiluminescence that occurred over the ensuing 5 minutes in response to addition of 100 µmol/L NADPH was recorded. In preliminary experiments, homogenates alone without addition of NADPH gave only minimal signals. Furthermore, NADPH did not evoke lucigenin chemiluminescence in the absence of homogenate. In some experiments, 30 U/mL of HB-SOD was added to determine the SOD-inhibitable activity. This is a recombinant form of SOD, which contains a heparin-binding domain that permits close association of the SOD with cell membranes, obviating electrostatic repulsion of SOD.

Measurement of Cytochrome Content
Aortas were removed, cleaned of periadventitial tissue, flushed with ice-cold phosphate-buffered saline, and immediately frozen in liquid nitrogen. Vessels were transferred to lysis buffer (1 mL per artery) containing 20 mmol/L monobasic potassium phosphate (pH 7.0), 1 mmol/L EGTA, 10 µg/mL aprotinin, 0.5 µg/mL leupeptin, 0.7 µg/mL pepstatin, and 0.5 mmol/L phenylmethane sulfonylfluoride and homogenized on ice. Cellular debris was pelleted by centrifugation at 250g, and the supernatant was recentrifuged at 29 000g at 4°C for 20 minutes. The resulting membrane pellet was dissolved in ice-cold 100 mmol/L sodium phosphate buffer (pH 7.2) containing protease inhibitors, homogenized, and incubated with agitation with 2% reduced Triton X-100 for 1 hour at 4°C. The insoluble fraction was separated by centrifugation (29 000g, 20 minutes, 4°C), and the supernatant was recentrifuged at 117 000g for 70 minutes at 4°C. The resulting supernatant, containing solubilized cytochromes, had a final protein concentration of 0.4 mg/mL.

Reduced minus oxidized difference spectra were recorded on 1-mL samples with a dual beam-scanning spectrophotometer (Perkin-Elmer Lambda 2S) as described by Jones et al.¹³ The baseline (oxidized) spectrum was recorded over 520 to 570 nm, and then a few grains of sodium dithionite were added to the sample cuvette for 1 minute to generate reduced cytochromes. This spectrum was recorded after 1 minute. The oxidized spectrum was subtracted electronically from the reduced spectrum. Peak absorbance (553 nm) corresponded to the absorbance of the cytochrome that is p22phox-based, as determined using transfection of antisense p22phox in VSMCs.¹² The correlation of changes in absorbance with changes in protein concentration was established using a standard curve generated with serial dilutions of VSMC homogenate. Data are expressed as arbitrary units, since the precise content of this cytochrome in VSMCs is not known.

In Situ Hybridization
In situ hybridization was performed on sections of aorta from Ang II–infused rats, as previously described,¹⁵ using p22phox riboprobes transcribed with [³⁵S]UTP. A 0.8-kb Sal I fragment or an 0.8-kb BamHI fragment of rat p22phox subcloned into pSPORT was prepared for antisense or sense riboprobes, respectively. Cryosections of aorta were pretreated with paraformaldehyde and proteinase K and prehybridized in 100 µL hybridization buffer (50% formamide, 0.3 mol/L NaCl, 20 mmol/L Tris [pH 8.0], 5 mmol/L EDTA, 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin, 10% dextran sulfate, and 10 mmol/L dithiothreitol) at 42°C. Serial sections were hybridized with 6x10⁵ cpm of ³⁵S-labeled riboprobes at 55°C. After hybridization, the sections were washed with 2x SSC (1x SSC: 150 mmol/L NaCl and 15 mmol/L sodium citrate, pH 7.0) containing 10 mmol/L ß-mercaptoethanol and 1 mmol/L EDTA, treated with RNase A, and washed in the same buffer, followed by a high-stringency wash in 0.1x SSC with 10 mmol/L ß-mercaptoethanol and 1 mmol/L EDTA at 55°C. The slides were then washed in 0.5x SSC and dehydrated in graded alcohols containing 0.3 mol/L NH₄Ac. The sections were dried, coated with NTB2 nuclear track emulsion, and exposed in the dark at 4°C for 4 to 12 weeks. After development, sections were counterstained with hematoxylin and eosin to aid in cell identification.

Statistical Analysis
All data are given as mean±SEM. Statistical significance was determined using one-way ANOVA. Means were considered significantly different at values of P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of Ang II Infusion on Systolic Blood Pressure
The effect of Ang II infusion on systolic blood pressure 1, 3, 5, 8, and 14 days after pump implantation is shown in Table 1. A significant increase in blood pressure was first detectable 3 days after pump implantation and was maintained for up to 14 days.

View this table: [in this window] [in a new window]   Table 1. Effect of Ang II Infusion on Systolic Blood Pressure

p22phox Expression in Rat Aorta
p22phox mRNA levels were increased 3 days after starting Ang II infusion and reached a maximum at 5 days after surgery (338±41% of control levels, n=7) (Figs 1 and 2). The onset of this increase in mRNA expression correlated well with the onset of increased blood pressure. p22phox mRNA levels in sham-operated rats were unchanged (Fig 1). Furthermore, there was no difference in the mRNA level between sham-operated rats and rats that did not undergo an operation (data not shown).

View larger version (30K): [in this window] [in a new window]   Figure 1. p22phox mRNA expression in aorta during Ang II infusion (AII). Northern blot analysis of p22phox mRNA expression was performed 1, 3, 5, 8, and 14 days after the start of AII. The top panel shows a representative Northern blot, with the p22phox mRNA band at 0.8 kb. Ethidium bromide staining of the 28S ribosomal RNA after blotting is shown in the lower panel. This was used to normalize small differences in loading of total RNA or the efficiency of mRNA transfer to the membrane in each lane. S indicates sham operation.

In vascular cells in which p22phox expression is abolished by antisense transfection, expression of the cytochrome with peak absorbance at 553 nm is also abolished.¹² Measurement of the content of this cytochrome thus provides information about the expression of the oxidase protein. Cytochrome expression in sham-operated rats measured by spectral analysis is shown in Fig 3. In rats treated with Ang II for 5 days, cytochrome content was 2.0±0.5-fold higher than in sham-operated rats.

View larger version (24K): [in this window] [in a new window]   Figure 3. Regulation of vascular NADPH/NADH oxidase cytochrome expression in rat aorta. Aortas were removed, and cytochromes were solubilized from the membrane fraction. Cytochrome content was measured spectrophotometrically. The inset shows the reduced-oxidized difference spectra in samples prepared from sham-operated rats. Bars indicate the relative levels of the cytochrome in sham-operated animals and in animals infused with Ang II for 5 days. Data are mean±SE from three independent experiments. *P<.05 vs sham-operated rats.

NADPH Oxidase Activity in Ang II–Infused Rats
To determine whether the observed increase in p22phox mRNA expression was accompanied by an increase in NADPH/NADH oxidase activity, we measured NADPH-specific ·O₂^- production after Ang II infusion. As shown in Fig 2, Ang II–infused rats showed a significant increase in ·O₂^- production between 3 and 7 days compared with sham-operated rats, suggesting that the increase in p22phox mRNA is accompanied by an increase in the functional expression of the enzyme. As expected, a recombinant form of SOD, HB-SOD, completely inhibited NADPH oxidase activity (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 2. p22phox mRNA expression and NADPH oxidase activity in aortic tissue after Ang II infusion. Rats were infused with Ang II for the indicated times and were killed; their aortas were removed for measurement of p22phox mRNA (left ordinate) or NADPH oxidase activity (right ordinate). Each point represents the mean±SE of 3 to 10 determinations. *P<.05 vs sham-operated rats; **P<.01 vs sham-operated rats.

Effect of Losartan and Hydralazine on p22phox mRNA Levels and NADPH Oxidase Activity
To determine the relative contributions of Ang II and blood pressure to the increase in p22phox mRNA and NADPH/NADH oxidase activity, we used two separate antihypertensive agents to lower blood pressure. Losartan (25 mg/kg per day), an AT₁ receptor blocker, and hydralazine (15 mg/kg per day), an antihypertensive agent that is reported to act as a direct smooth muscle relaxant, were administered in the drinking water to Ang II infusion pump–implanted rats. After 5 days, both losartan and hydralazine decreased blood pressure to near control levels (124±3 and 138±6 mm Hg, respectively [Table 2]). Both losartan and hydralazine inhibited the Ang II–induced p22phox mRNA upregulation to levels equivalent to or below that of sham-operated rats (Fig 4). NADPH/NADH oxidase activity was also inhibited (Fig 4).

View this table: [in this window] [in a new window]   Table 2. Effect of Antihypertensive Treatments on Systolic Blood Pressure

View larger version (25K): [in this window] [in a new window]   Figure 4. p22phox mRNA expression and NADPH oxidase activity in aortic tissue after Ang II infusion in the presence and absence of antihypertensive agents. NADPH oxidase activity and p22phox mRNA levels were measured as described in "Materials and Methods." Some rats received losartan (25 mg/kg per day) or hydralazine (15 mg/kg per day) in their drinking water beginning 2 days before pump implantation. Bars represent p22phox mRNA expression (left) and NADPH oxidase activity (right) in sham-operated, Ang II–infused, losartan-treated, and hydralazine-treated rats. Each experiment was repeated 3 to 10 times. *P<.01 vs sham-operated rats.

Effect of SOD Infusion on Blood Pressure and p22phox Expression
To determine the relationship between ·O₂^- production, p22phox expression, and the development of hypertension, we infused rats with a recombinant form of SOD, HB-SOD, that is targeted to the vasculature. Continuous infusion of HB-SOD significantly reduced the Ang II–induced increase in blood pressure (Ang II, 192±3 mm Hg; Ang II+HB-SOD, 130±3 mm Hg; n=7; P<.05). It also markedly attenuated the increase in p22phox mRNA induced by 5 days of Ang II infusion (Fig 5). Infusion of HB-SOD alone caused a small increase in basal p22phox expression (data not shown).

View larger version (33K): [in this window] [in a new window]   Figure 5. Effect of SOD infusion on p22phox mRNA expression. Rats were treated with HB-SOD or vehicle for 3 days before the start of Ang II infusion. Ang II was then infused for 5 days, and aortas were removed for isolation of RNA. The top panel is a representative Northern blot showing the band for p22phox mRNA. The 28S ribosomal RNA visualized with ethidium bromide was used for normalization. The bar graph summarizes data obtained from seven rats in each group, expressed as the percent change over the levels in sham-operated rats (mean±SEM). *P<.01 vs sham-operated rats.

In Situ Hybridization of p22phox mRNA
Because p22phox is highly expressed in neutrophils and is found in several types of vascular cells, we used in situ hybridization to determine which cell types express this mRNA in Ang II–infused rats. As shown in Fig 6A, p22phox mRNA is expressed both in medial smooth muscle cells and in the adventitia. Sense riboprobes did not detect p22phox mRNA expression (Fig 6B). Staining of adjacent sections for the presence of neutrophils showed no correlation of neutrophil accumulation and p22phox localization (data not shown).

View larger version (114K): [in this window] [in a new window]   Figure 6. Localization of p22phox mRNA in rat aorta by in situ hybridization. Serial sections from an aorta of Ang II–infused rats were hybridized with antisense (A) or sense (B) 35S-labeled riboprobes specific for p22phox. A, Medial smooth muscle and adventitia show positive hybridization with the antisense probe. B, Sense control shows essentially no hybridization within the arterial wall. Magnification x100; exposure time, 14 weeks.

	Discussion

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Recent work has raised the possibility that ·O₂^- may be involved in the pathogenesis of hypertension.³ We⁵ and others⁸ have identified a membrane-associated NADPH/NADH oxidase in vascular cells responsible for the majority of ·O₂^- production by VSMCs. Studies from our laboratories have shown that it is hormone sensitive.^{5 14} This oxidase is similar, but not identical, to that found in neutrophils and, in particular, shares the cytochrome b₅₅₈ -subunit, p22phox.¹¹ In the present study, we demonstrate for the first time that the mRNA for this protein is expressed in medial smooth muscle in vivo and that it is upregulated in the aortas of rats made hypertensive by Ang II infusion. This increase is accompanied by an increase in expression of the vascular NADPH/NADH oxidase cytochrome and NADPH oxidase activity. The fact that HB-SOD partially inhibits the increase in blood pressure in response to Ang II infusion suggests that this oxidase system may have a functional role in types of hypertension in which Ang II levels are increased.

Fibroblasts, mesangial cells, and VMSCs all have been shown to generate ·O₂^- via an NADPH/NADH oxidase similar to that in phagocytes.^{5 6 16} Although the NADPH/NADH oxidase system in rat VSMCs shares many features of the phagocyte NADPH oxidase, including its nonmitochondrial location, its activation by phosphatidic acid, and its sensitivity to diphenylene iodinium, it differs from the phagocyte NADPH system in its kinetic properties, its physiological function, and possibly its subunit structure. NADPH/NADH oxidase activation in rat VSMCs, as well as fibroblasts and mesangial cells, occurs over a period of hours,^{5 6} whereas ·O₂^- generation in phagocytes begins within minutes.¹⁷ Furthermore, the phagocytic oxidase produces much more ·O₂^- than does the vascular oxidase.^{5 17} The physiological function of the ·O₂^- in smooth muscle cells is also likely to be different from that of phagocytes. In phagocytes, ·O₂^- is bactericidal, whereas in VSMCs, it participates in other processes, such as regulation of nitric oxide activity, hypertrophy, and modulation of redox state.^{5 12 18 19} With regard to the subunit structure of the oxidase, it has been shown that the fibroblast cytochrome b₅₅₈ is immunologically distinct from phagocytes and that the mesangial cell cytochrome b₅₅₈ is antigenically related, but not identical, to that of phagocytes.^{16 20} Although VSMC p22phox is highly homologous to the neutrophil protein, gp91phox has not been identified in nonphagocytic cells. However, p22phox has been found in fibroblasts and mesangial cells^{13 21} and appears to lead to expression of a cytochrome b₅₅₈ even in the absence of gp91phox, calling into question the suggestion that one subunit of cytochrome b₅₅₈ cannot be expressed without the other.²² In VSMCs, expression of p22phox is also accompanied by the presence of a spectrophotometrically identifiable cytochrome (peak, 553 nm) and a functional NADPH oxidase activity (Figs 2 and 3 and Reference 12). It is of note that depletion of p22phox by antisense transfection of VSMCs markedly attenuates NADPH and NADH-driven ·O₂^- formation,¹² providing definitive evidence that this subunit is an integral part of the vascular NADPH/NADH oxidase.

In the present study, we used NADPH oxidase activity and spectrophotometric expression of the cytochrome as a measure of functional enzyme expression. Antibodies raised against human neutrophil oxidase components (including p22phox, p47phox, and p67phox) did not cross-react with rat aortic tissues or rat VSMCs (authors' unpublished data, 1996); therefore, we were unable to measure protein immunologically. However, the changes in p22phox mRNA expression in Ang II–induced hypertension were accompanied by parallel changes in NADPH oxidase activity and cytochrome expression, as demonstrated spectrophotometrically, suggesting that the increased mRNA is in fact accompanied by expression of the protein. Thus, it seems that in this model of hypertension, the increase in NADPH/NADH oxidase activity is due, at least in part, to upregulation of one or several components of this oxidase system at the mRNA level.

Because neutrophils and macrophages have a high level of NADPH oxidase activity,¹⁷ we considered the possibility that the increase in p22phox mRNA expression in hypertensive aortas might be due to migration of inflammatory cells into the vessel wall. By immunocytochemistry, no inflammatory cells were noted in the medial layer, and inflammatory cells in the adventitia did not correlate with p22phox mRNA expression. However, to minimize any possible contribution of these cells in our Northern blot experiments, we carefully removed as much of the periadventitial tissue as possible before homogenizing the aortic tissue.

The relationship between Ang II, blood pressure, and p22phox mRNA expression/NADPH oxidase activity is complex. Blood pressure and mRNA expression both began to increase simultaneously at 3 days after implantation. NADPH oxidase activity was also increased by day 3, suggesting that alteration in the oxidative state of the vessel wall is a relatively early response to high levels of Ang II. This early increase in oxidase activity may result from activation of existing enzyme, but the later increase correlates well with increased p22phox mRNA expression. It is possible that the increase in blood pressure is the stimulus that induces p22phox mRNA expression, but this seems unlikely on the basis of prior studies in which we have shown that norepinephrine-induced hypertension does not increase NADPH/NADH oxidase activity.¹⁴ It is perhaps more likely that a specific Ang II–mediated increase in NADPH/NADH oxidase activity is contributing to increased blood pressure. Although we cannot fully distinguish between these possibilities, the experiments using antihypertensive interventions provide some insight. Losartan blocks both the cellular effects of Ang II and the rise in blood pressure, whereas hydralazine has been traditionally viewed as countering only the effects of blood pressure elevation. Both of these agents decrease p22phox mRNA expression and NADPH/NADH oxidase activity, implying that blood pressure itself might regulate cytochrome expression in the setting of elevated Ang II. However, recent evidence suggests that hydralazine also blocks activation of the oxidase,²³ raising the possibility that both drugs prevent Ang II stimulation of the NADPH/NADH oxidase. Even if p22phox mRNA expression and NADPH/NADH oxidase activity are a consequence, rather than a cause, of elevated blood pressure, the excess generation of ·O₂^- may explain the increased risk of atherosclerosis in patients with hypertension,²⁴ given the reported role of reactive oxygen species in atherogenesis.¹

Upregulation of p22phox mRNA and the increase in NADPH/NADH oxidase activity correlate well with the development of increased blood pressure. However, p22phox mRNA expression starts to decrease 8 to 14 days after pump implantation, whereas blood pressure remains elevated. By 14 days, there is a significant increase in wall thickness (Reference 10 and Q. Capers and W.R. Taylor, unpublished data, 1996), which may play a role in sustaining hypertension. It is also possible that early activation of the NADPH/NADH oxidase stimulates redox-sensitive pathways that then sustain hypertension.¹ This suggests that the ·O₂^-, which is produced as a result of oxidase upregulation, may contribute to the development of hypertension. This possibility is supported by the observation that SOD infusion nearly abolished the Ang II–induced rise in blood pressure.

The experiments with SOD raise several issues about the relationship between blood pressure regulation and oxygen-derived free radicals. SOD blocks both the increase in blood pressure and the accumulation of ·O₂^- in the vessel wall. The effect on blood pressure may be related to an increased half-life of nitric oxide by the removal of inactivating ·O₂^-.¹⁸ The fact that SOD attenuated the increase in p22phox mRNA expression in hypertensive animals suggests that ·O₂^- itself may regulate expression of p22phox, although this prospect needs to be addressed directly. Thus, it is possible that in hypertension there is an imbalance in the mechanisms regulating ·O₂^- production, so that a self-perpetuating system develops in which blood pressure increases p22phox mRNA expression and NADPH oxidase activity, resulting in supraphysiological production of ·O₂^-, which contributes to hypertension by inactivating NO¹⁸ and increasing vascular hypertrophy.⁵

The present experiments provide one of the first demonstrations that p22phox expression can be regulated. In neutrophils, interferon- and lipopolysaccharide induce ·O₂^- generation but have no effect on p22phox expression.^{10 25} It is interesting that upregulation of this component in the present study was reflected in increased NADPH oxidase activity, suggesting that levels of this subunit are limiting, that all subunits of the oxidase are upregulated in this form of hypertension, or that regulation of p22phox promotes an increase in expression of other subunits. Resolution of this issue awaits identification of the other components of the smooth muscle oxidase.

In conclusion, we have shown that p22phox mRNA is expressed in the medial layer of the rat aorta. In the Ang II infusion model of hypertension, expression of this mRNA is markedly increased, and this upregulation is accompanied by an increase in activity of the NADPH oxidase. These results suggest that the NADPH oxidase system may be causally or consequentially associated with hypertension in vivo and provide a potential biological basis for the increased risk of atherosclerosis associated with hypertension.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II HB-SOD = heparin-binding SOD SOD = superoxide dismutase VSMC = vascular smooth muscle cell

	Acknowledgments

 
This study was supported by National Institutes of Health grant HL-38206. We are grateful to Dr Bruce Freeman for his generous gift of HB-SOD and to Dr R.D. Smith for his gift of losartan. We thank Drs A. Maziar Zafari and Gilles De Keulenaer for helpful discussions, Dr Dale Edmondson for his help developing the cytochrome content assay, and Barbara Merchant-Bailey for editorial assistance.

	Footnotes

 
*Dr. Fukui and Dr. Ishizaka contributed equally to this work.

Received October 11, 1995; accepted October 17, 1996.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
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