A more recent version of this article appeared on August 31, 2001

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© 2001 American Heart Association, Inc.

Article

Novel Competitive Inhibitor of NAD(P)H Oxidase Assembly Attenuates Vascular O₂^- and Systolic Blood Pressure in Mice

F. E. Rey, M. E. Cifuentes, A. Kiarash, M. T. Quinn P. J. Pagano

From the Hypertension and Vascular Research Division (F.E.R., M.E.C., A.K., P.J.P.), Henry Ford Hospital, Detroit, Mich; and Veterinary Molecular Biology Laboratory (M.T.Q.), Montana State University, Bozeman, Mont.

Correspondence to Patrick J. Pagano, PhD, Hypertension & Vascular Research Division, Room 7044, E&R Bldg, Henry Ford Hospital, 2799 W Grand Blvd, Detroit, MI 48202-2689. E-mail ppagano1{at}hfhs.org

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We previously reported enhanced expression of the p67^phox and gp91^phox components of NAD(P)H oxidase in angiotensin (Ang) II–induced hypertension, suggesting de novo assembly in response to Ang II. To examine the direct involvement of NAD(P)H oxidases in Ang II–induced O₂^- production, we designed a chimeric peptide that inhibits p47^phox association with gp91^phox in NAD(P)H oxidase (gp91ds-tat). This was achieved by linking a 9-amino acid peptide (aa) derived from HIV-coat protein (tat) to a 9-aa sequence of gp91^phox (known to interact with p47^phox). As a control, we constructed a chimera containing tat and a scrambled gp91 sequence (scramb-tat). We found that gp91ds-tat decreased O₂^- levels in aortic rings treated with Ang II (10 pmol/L) but had no effect on either the O₂^--generating enzyme xanthine oxidase or potassium superoxide–generated O₂^-. We infused vehicle, Ang II (0.75 mg · kg^-1 · d^-1), Ang II+gp91ds-tat (10 mg · kg^-1 · d^-1), or Ang II+scramb-tat intraperitoneally in C57Bl/6 mice and measured systolic blood pressure (SBP) on days 0, 3, 5, and 7 of infusion. SBP increased by day 3 in mice given Ang II and Ang II+scramb-tat but was significantly lower with Ang II+gp91-tat. On day 7, SBP was still significantly inhibited in mice given Ang II+gp91ds-tat, whereas Ang II–induced O₂^- production was inhibited throughout the aorta as detected by dihydroethidium staining, consistent with the ability of this inhibitor to block the various vascular NAD(P)H oxidase isoforms. These data support the hypothesis that inhibition of the interaction of p47^phox and gp91^phox (or its homologues) can block O₂^- production and attenuate blood pressure elevation in mice.

Key Words: superoxide • angiotensin II • NAD(P)H oxidase • gp91^phox • p47^phox

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Superoxide anion (O₂^-) can interfere with nitric oxide (NO)–dependent vasodilatation^1–4 and participate in endothelium-dependent constriction.^5,6 O₂^- has also been shown to affect the sensitivity of blood vessels to vasodilators.⁷ Numerous reports demonstrate that superoxide dismutase (SOD) can improve relaxation of blood vessels.^1,3,4 We and others have suggested that NAD(P)H oxidase–derived O₂^- inhibits NO-dependent relaxation,^8–10 and NAD(P)H oxidase–derived O₂^- has been implicated in angiotensin (Ang) II–induced blood pressure elevation.^8,11,12

A number of studies have shown that a phagocyte-like NAD(P)H oxidase is the major source of O₂^- in vascular tissue.^8,13,14 Recent studies show that Ang II increases mRNA levels of p22^phox and p67^phox15,16 and can also stimulate NAD(P)H oxidase O₂^- production by neutrophils¹⁷ as well as by the vascular endothelium, medial smooth muscle cells, and adventitial fibroblasts.^{9,15,16,18,19} It is well known that activation of NAD(P)H oxidase in neutrophils is triggered via protein kinase C (PKC)–mediated phosphorylation of cytosolic p47^phox, which then binds to membrane-associated gp91^phox.²⁰ Recently, a similar process has been inferred in the vasculature, since it was shown in a model of renovascular hypertension (2 kidneys, 1 clip) that calphostin C, an inhibitor of PKC, decreased O₂^- levels in aortas from hypertensive animals,²¹ suggesting that the mechanism of activation is similar between neutrophil and vascular NAD(P)H oxidase. Moreover, p47^phox translocates to the membrane of endothelial and smooth muscle cells upon stimulation with phorbol esters and thrombin, respectively.^22–24 However, it is unclear whether gp91^phox, its smooth muscle homologue nox1-containing NAD(P)H oxidase,²⁵ or nox4^26,27 mediates Ang II–dependent increases in O₂^- in the vasculature and participates in the development of hypertension.

Although there is evidence linking NAD(P)H oxidase and hypertension, there is a lack of effective inhibitors targeting this oxidase that do not inhibit other flavin-dependent enzymes. For this reason, we designed chimeric peptides that would interfere with the assembly of vascular NAD(P)H oxidase components. In human neutrophils, small peptide sequences of gp91^phox, which are involved in the binding of gp91^phox to p47^phox, inhibit O₂^- formation in cell-free assays.^28,29 We selected the sequence found to be most potent in cell-free human neutrophil assays and then determined the corresponding sequence from the gp91^phox mouse clone,³⁰ calling it gp91ds (gp91 docking sequence). This represents a 1-amino acid difference between the human and mouse neutrophil sequence, a substitution of isoleucine for valine at amino acid 89. Using the LALIGN server, we recently found that homologous sequences exist in nox1²⁵ and nox4²⁶ that correspond to the site of p47^phox binding, and thus we expect the peptide to be useful in blocking the involvement of oxidases containing these homologues. Indeed, since previous reports show a functional requirement of p47^phox translocation to plasma membranes in smooth muscle cells,²⁴ which have been shown to contain primarily nox1 and nox4,³¹ interaction of p47^phox with anchoring gp91^phox-like components appears to be fundamental to this family of enzymes. As we aimed to deliver this peptide either to the whole animal or intact vessels, we linked it to a specific 9-amino acid peptide of HIV viral coats (HIV-tat), which is known to be internalized by all cells³² and was shown to deliver conjugated proteins after intravenous injection³³ (gp91ds-tat). As a control, we scrambled the 9-amino acid gp91^phox sequence in a way that generated the lowest number of hits when matched against the GenBank database and linked it to the 9-amino acid tat peptide in the same fashion (scramb-tat). In vitro, gp91ds-tat was found to be effective at blocking Ang II–induced O₂^- levels in the mouse aorta. To test the efficacy of this NAD(P)H oxidase inhibitor in vivo, we coinfused the chimeric peptide with Ang II and examined its effects on blood pressure elevation and vascular O₂^-.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lucigenin and diethyldithiocarbamate (DDC) (Sigma) were solubilized in physiological buffer; dimethylsulfoxide 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron; Sigma) was solubilized in H₂O. Ang II was obtained from Sigma and diluted in 0.9% saline with 0.01 N acetic acid. Chimeric peptides (patent pending) were synthesized by the Protein Chemistry Facility of Tufts University (Boston, Mass) and diluted in solutions containing Ang II.

The sequence of gp91ds-tat (patent pending) is as follows:

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The sequence of scramb-tat (patent pending) is as follows:

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Peptides were synthesized with an ABI 431A peptide synthesizer using FMOC amino acids and HTBU activator. The purity of these preparations was 70%, with the balance being deletions and chain terminations of the full-length peptide. Further attempts at purification using reverse-phase C18 column chromatography did not result in substantially greater purity of the peptide or biological activity and also decreased yield considerably.

Animals and Systolic Blood Pressure (SBP) Measurements
C57Bl/6Tac male mice 8 to 9 weeks old were purchased from Taconic Farms (Germantown, NY), and SBP was measured as described in the online data supplement.

Surgery and Peptide Infusions
Live animals were handled in accordance with the NIH Guidelines for the Care and Use of Experimental Animals, and protocols were approved by the Henry Ford Hospital Committee for Care and Use of Animals. Alzet osmotic minipumps containing either vehicle (0.01 N acetic acid in saline solution), Ang II (0.75 mg · kg^-1 · d^-1), Ang II+gp91ds-tat (10 mg · kg^-1 · d^-1), or Ang II+scramb-tat (10 mg · kg^-1 · d^-1) were implanted intraperitoneally under sterile conditions. Seven days later, animals were killed and the thoracic and abdominal aortas were removed.

Ex Vivo Measurement of O₂^-
Aortas from mice infused with Ang II, Ang II+gp91ds-tat, or Ang II+scramb-tat were cleaned of adipose tissue and cut into rings and incubated for 30 minutes in modified Krebs-HEPES buffer (pH 7.4) at 37°C in the presence of DDC (10 mmol/L) to inhibit endogenous Cu/Zn SOD as described previously.¹² Luminescence measurements were integrated for 30-second periods and the cycle repeated 9 times, then averaged. A standard curve was obtained as described previously¹² to express values in nmol O₂^-/min per mg of tissue.

Dihydroethidium (HE) Staining
HE was used to evaluate in situ production of superoxide.³⁴ Unfixed frozen ring segments from vehicle, Ang II, Ang II+gp91ds-tat, and Ang II+scramb-tat–treated mice were cut into 30-µm-thick sections and placed on a glass slide. HE (2 µmol/L) was topically applied to each tissue section. Images were obtained with a Bio-Rad MRC-1024 laser scanning confocal microscope.

NAD(P)H Oxidase Inhibition In Vitro
Aortas were removed, cleaned as described above, and preincubated for 30 minutes in Krebs-HEPES buffer with either gp91ds-tat (50 µmol/L), tat, or vehicle at 37°C. Ang II (10 pmol/L) or vehicle was added and the tube incubated for 3 hours. The same procedures as for the ex vivo measurements were followed except that the lucigenin concentration was 25 µmol/L.

NAD(P)H Oxidase Activity in Aortic Fibroblasts
We tested the capacity of gp91ds-tat to inhibit NADPH-dependent oxidase activity in membranes from cultured rat aortic adventitial fibroblasts. Cells were preincubated with vehicle or gp91ds-tat (30 minutes) and then with vehicle, Ang II (10 nmol/L), or Ang II+gp91ds-tat (50 µmol/L) for 3 hours. Oxidase activity is expressed as a percentage relative to activity from vehicle-treated cells.

Effect of gp91ds-tat on Human Neutrophil O₂^- Production
The effectiveness of gp91ds-tat to inhibit O₂^- production in intact neutrophils was measured using a standard cytochrome c–based assay as described previously.³⁵

Tests of gp91ds-tat Specificity
To confirm the specificity of gp91ds-tat, we tested its effect on the potassium superoxide (KO₂)–generated O₂^-. Formation of O₂^- anion by KO₂ was determined in the presence of vehicle or gp91ds-tat. We also tested the effect of gp91ds-tat on xanthine oxidase (XO)/hypoxanthine–derived O₂^-. XO (0.1 U/mL) was incubated for 3 hours at room temperature with vehicle or gp91ds-tat (50 µmol/L). Measurements were made using 5 µmol/L lucigenin.

Statistical Analysis
Data are expressed as mean±SEM, with n as the number of animals for each experiment. The significance of point differences in O₂^- generation in vitro was analyzed by Student’s t test. Differences in SBP were analyzed by ANOVA followed by Holm’s method for multiple comparisons. Differences in ex vivo O₂^- production were analyzed by ANOVA followed by Tukey’s comparison. P<0.05 was considered significant.

An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In Vitro Measurements of O₂^-
We first tested the ability of gp91ds-tat to inhibit vascular O₂^- in vitro. Ang II (10 pmol/L) increased aortic O₂^- 2.5-fold compared with vehicle control. Preincubation of aortic rings with gp91ds-tat (50 µmol/L) for 30 minutes completely blocked Ang II–induced O₂^- production (n=8). In contrast, preincubation of rings for 30 minutes with tat alone did not inhibit Ang II–induced O₂^-, consistent with gp91ds-tat inhibiting aortic oxidase and O₂^- generation (Figure 1). To confirm that gp91ds-tat was capable of inhibiting subcellular oxidase activity, quiescent rat aortic fibroblasts treated with Ang II demonstrated NAD(P)H oxidase activity that was 165±35% of vehicle control. This increase was substantially lower than we expected and previously reported for rabbit fibroblasts. Nonetheless, this activity was reduced to 80±19% of vehicle control activity by coincubation with gp91ds-tat (n=5).

View larger version (37K): [in this window] [in a new window]   Figure 1. In vitro measurements of O2- in mouse aortic rings treated (or not) with a competitive inhibitor of NAD(P)H oxidase. Aortas were preincubated for 30 minutes with vehicle, gp91ds-tat, or tat followed by 3 hours of incubation with vehicle or Ang II (10 pmol/L). Data are expressed as delta of units of chemiluminescence/min per mg tissue before and after Tiron measured by lucigenin-enhanced chemiluminescence (25 µmol/L) and are the mean±SEM of 8 aortas. *Statistical significance by unpaired t test vs control, P<0.05; **significance by unpaired t test vs Ang II treatment, P<0.05.

We further examined the specificity of gp91ds-tat to inhibit NAD(P)H oxidase by two means. First, we examined whether gp91ds-tat could directly scavenge O₂^- produced by an inorganic source. KO₂ is known to react in aqueous solutions, releasing O₂^-.³⁶ Production of O₂^- by KO₂ was measured in the presence of vehicle, gp91ds-tat (50 µmol/L), or SOD (150 U/mL). SOD significantly reduced O₂^- detection (n=5), but no effect was observed in the presence of gp91ds-tat (n=6) (Figure 2A).

View larger version (32K): [in this window] [in a new window]   Figure 2. Lack of effect of gp91ds-tat on O2- production by KO2 and XO. A, Effect of 50 µmol/L gp91ds-tat and SOD on O2- levels generated by the aqueous reaction of KO2. B, Effects of gp91ds-tat on O2- production by XO (0.002 U/mL)/hypoxanthine (1 mmol/L). Data are the mean±SEM. XO was incubated with 50 µmol/L gp91ds-tat or vehicle for 3 hours before and during measurement of O2-. *Statistical significance by unpaired t test vs vehicle, P<0.05.

We also tested whether the chimeric peptide could inhibit another prevalent vascular enzymatic O₂^- source. XO (0.1 U/mL) was incubated for 3 hours at room temperature with vehicle or gp91ds-tat (50 µmol/L). An aliquot of XO was added to tubes (dilution 1:50) containing lucigenin (5 µmol/L) and gp91ds-tat (50 µmol/L). gp91ds-tat did not affect O₂^- production by XO (n=4) (Figure 2B), suggesting that gp91ds-tat is unable to directly inhibit this enzyme and corroborating its inability to directly scavenge O₂^-.

In Vivo Effects of gp91ds-tat
To test the ability of the inhibitors to decrease vascular O₂^- and blood pressure in vivo, we infused mice with Ang II in the presence and absence of gp91ds-tat or scramb-tat. As expected, Ang II infusion significantly increased total aortic O₂^- ex vivo compared with sham (n=6), and cotreatment with gp91ds-tat abolished the increase produced by Ang II (n=5). On the contrary, scramb-tat was ineffective at inhibiting Ang II–induced O₂^- formation (n=4) (Figure 3).

View larger version (32K): [in this window] [in a new window]   Figure 3. Ex vivo measurements of O2- in mouse aortic rings. Aortas were removed from mice treated for 7 days with vehicle (n=5), Ang II (n=5), Ang II+gp91ds-tat (n=5), or Ang II+scramb- tat (n=4), equilibrated, and O2- measured by chemiluminescence. Data are the mean±SEM. *Statistical significance by ANOVA (Tukey’s comparison) vs sham-operated controls, P<0.05; **significance vs Ang II treatment, P<0.05.

HE Staining
To further test the effect of gp91ds-tat on vascular cell O₂^- generation, HE was compared in aortas from the various treatment groups. Ang II treatment significantly increased the fluorescent signal in the three vascular layers compared with vehicle, indicative of an increase in O₂^- in all segments of the aorta. This increase was inhibited throughout the aorta by cotreatment with gp91ds-tat but not scramb-tat (Figure 4).

View larger version (11K): [in this window] [in a new window]   Figure 4. In situ detection of superoxide using HE staining from representative vehicle- (A), Ang II– (B), Ang II+gp91ds-tat– (C), and Ang II+scramb-tat– (D) treated mice. Conversion of HE by O2- to ethidium results in red nuclear fluorescence. Unfixed frozen aortic rings (30 µm) were incubated with HE (2 µmol/L) and visualized by confocal microscopy. Data are representative of 3 experiments. L indicates lumen; M, media; and A, adventitia.

Effect of gp91ds-tat on SBP
Ang II infusion (0.75 mg · kg^-1 · d^-1, IP) significantly increased SBP compared with vehicle (Figure 5). Coinfusion with gp91ds-tat significantly attenuated SBP at all time points after implantation compared with Ang II+scramb-tat or Ang II alone (Figures 5A and 5B, respectively) and reduced the increase caused by Ang II alone by 44% on day 7 (P=0.009). On the other hand, coinfusion of scramb-tat did not affect SBP compared with Ang II alone.

View larger version (17K): [in this window] [in a new window]   Figure 5. SBP was measured by tail cuff on day 0 (before surgery) and on days 3, 5, and 7 after implantation of osmotic minipumps. A, Comparisons of data from vehicle- (sham), Ang II+gp91ds-tat–, and Ang II+scramb-tat–treated mice. B, Comparisons of vehicle (sham), Ang II, and Ang II+gp91ds-tat. Data are the mean±SEM. *Statistical significance between Ang II or Ang II+scramb-tat vs Ang II+gp91ds-tat at 3, 5, and 7 days by ANOVA; Holm’s method was used to adjust the significance level for multiple comparisons.

Effect of gp91ds-tat on O₂^- Production by Neutrophils
We tested the ability of gp91ds-tat to inhibit O₂^- production in neutrophils on activation with phorbol myristate acetate (PMA). Although the gp91ds sequence blocked neutrophil O₂^- by 80% in a cell-free assay,²⁸ O₂^- production was reduced only by 35% when the highest concentration of gp91ds-tat (100 µmol/L) was used in the cell-intact assay (Figure 6A; P<0.01; n=3). Scramb-tat did not significantly reduce neutrophil O₂^- production (Figure 6B).

View larger version (45K): [in this window] [in a new window]   Figure 6. Effect of gp91ds-tat and scramb-tat on O2- production by isolated intact human neutrophils. Preincubated neutrophils were treated with PMA (100 µg/mL), and O2- was measured by cytochrome c reduction. Data are the mean±SEM. *Statistical significance by unpaired t test, P<0.05; **P<0.01.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The results reported in the present study are evidence for direct involvement of NAD(P)H oxidases in Ang II–induced O₂^- and blood pressure elevation and provide a novel means of inhibiting NAD(P)H oxidase in whole-cell preparations and in vivo. Previous attempts at testing the contribution of NAD(P)H oxidase to increased O₂^- and blood pressure in vivo have been difficult because of the lack of specific and effective inhibitors of vascular NAD(P)H oxidases; for example, diphenylene iodonium, the classic inhibitor of NAD(P)H oxidase, also inhibits a variety of enzymes including XO³⁷ and NO synthases.³⁸ We previously detected phagocyte-like gp91^phox in aortas of rabbits^14,39 and rats¹⁰ and showed upregulation in response to Ang II in mice.¹² Others have demonstrated its homologues nox1 and nox4 in vascular cells²⁴; these appear to be related to gp91^phox at the N-terminal end, which contains regions that interact with p47^phox. Based on this knowledge, we developed a competitive antagonist of the interaction of gp91^phox and p47^phox and showed that it can suppress the induction of O₂^- by Ang II. Moreover, infusion of this novel cell-permeant NAD(P)H oxidase inhibitor reduced blood pressure elevation and aortic O₂^- in response to Ang II in vivo. The lack of any noticeable adverse reaction in these mice, as well as the fact that scramb-tat at the same concentration had no effect, indicated that the dose we used was safe in vivo. These data are consistent with the hypothesis that induction of vascular NAD(P)H oxidase–derived O₂^- contributes to blood pressure elevation.

We and others have reported finding four major NAD(P)H oxidase components, gp91^phox, p47^phox, p67^phox, and p22^phox, in the vascular wall^22,39,40 and observed upregulation of p22^phox, gp91^phox, and p67^phox by Ang II, concomitant with significant elevations in O₂^-.^12,15 In addition, p47^phox has been reported to be an essential component in the vasculature, since mice lacking this subunit showed lower levels of O₂^- compared with wild-type mice.⁴¹ These findings provided a further rationale for development of competitive inhibitors of gp91^phox and p47^phox interaction. In these studies, we developed a means to inhibit ongoing assembly of NAD(P)H oxidase in response to Ang II by interfering with the interaction of these two critical components. When cytosolic p47^phox is activated in neutrophils, it is phosphorylated, which enhances its interaction with p67^phox. This complex then translocates to the inner surface of the plasma membrane where p47^phox interacts with gp91^phox, which is necessary for full activation of the enzyme.²⁸ Evidence for this translocation has been reported for endothelial cells and smooth muscle cells.²² Thus gp91ds-tat was designed to inhibit the interaction of gp91^phox and p47^phox.

Other studies have suggested the use of a cell-permeant antimicrobial peptide called PR-39 to inhibit NAD(P)H oxidase assembly, but its intracellular target is not clear and its antimicrobial properties raise some concern.⁴² Apocynin is described as an effective inhibitor of phagocyte oxidase assembly that has recently been shown to inhibit endothelial cell O₂^-.²² However, the activity of apocynin reportedly requires its conversion by myeloperoxidase,⁴³ which is only present in myeloid cells.⁴⁴ Thus, it is unclear why apocynin is active in endothelial cells. Perhaps, analogous peroxidases subserve such activation in the endothelium. Incidentally, we have been unable to show that apocynin can attenuate O₂^- generation in rat aortic smooth muscle cells and fibroblasts (authors’ unpublished observations, 2001).

The peptide inhibitor sequence we chose (gp91ds) was found to be most potent compared with other related sequences in neutrophil cell–free assays, with an IC₅₀ of 1 to 3 µmol/L and an ability to block 80% of O₂^- production.^28,29 Although we have not obtained full dose-response curves for gp91ds-tat in vascular tissue, the concentration we used in our in vitro studies is consistent with the previously reported efficacy of gp91ds alone.²⁹ Moreover, the ineffectiveness of tat or scramb-tat precludes a nonspecific effect of cell membrane transmigration on O₂^- measurements. We also tested the hypothesis that gp91ds-tat could directly scavenge O₂^- or inhibit other enzymes. For this, we examined its effect on KO₂ and XO/hypoxanthine-generated O₂^- in vitro. In both cases, gp91ds-tat failed to alter O₂^- steady-state levels, indicating that it is not an O₂^- scavenger or an inhibitor of XO.

gp91ds-tat completely inhibited Ang II–induced aortic O₂^- both in vitro and in vivo as measured by two methods of O₂^- detection. Using chemiluminescence, we found that the Ang II–induced increase in whole aortic O₂^- levels was returned to control levels by gp91ds-tat. We also showed that Ang II–induced NAD(P)H oxidase activity in fibroblasts was reversed by gp91ds-tat confirming the ability of the peptide to specifically inhibit this oxidase. Ang II has been reported to significantly increase gp91^phox12 and nox1, a shorter homologue of gp91^phox involved in smooth muscle oxidase activity,²⁵ and antisense to nox1 can attenuate such an increase.³¹ Thus, we cannot exclude the possibility that gp91ds-tat is inhibiting oxidase isoforms containing both these homologues. Indeed, we showed that Ang II–induced HE staining was reduced by gp91ds-tat in all three vascular layers. Not only do these findings with HE confirm our results with chemiluminescence, but they also suggest that gp91ds-tat can inhibit the various vascular oxidase isoforms. When we searched the rat nox1 sequence LALIGN server (available at http://www.ch.embnet.org/software/LALIGN_form.html), we found a region of conserved homology with our inhibitory peptide (amino acids 86 to 94).²⁵ Moreover, renox (nox4), first reported in the kidney²⁶ and now shown to be expressed in smooth muscle cells,³¹ has substantial homology with the gp91ds peptide sequence (amino acids 91 to 98). However, Lassègue et al³¹ have shown that nox4 levels are decreased in response to Ang II, and thus it is unlikely that nox4 accounts for the increase in medial HE staining. The fact that HE staining was inhibited by gp91ds-tat in all vascular layers is consistent with its ability to inhibit at least 2 of the NAD(P)H oxidase isoforms (gp91^phox and nox1). Future studies will be necessary to directly examine the ability of this peptide to inhibit assembly of nox1 and nox4 with p47^phox.

gp91ds has been shown to effectively inhibit neutrophil oxidase assembly in a cell-free assay.^28,29 However, upon testing the effect of these compounds on O₂^- production by intact human neutrophils, we found lower inhibitory activity; that is, concentrations of gp91ds-tat up to 100 µmol/L were only capable of decreasing O₂^- by 35%. It is noteworthy that 50 µmol/L gp91ds-tat completely inhibited Ang II–induced aortic O₂^- but was only capable of inhibiting neutrophil activity by 24%. It is not clear why gp91ds-tat was more effective in inhibiting vascular cell NAD(P)H oxidase in these studies. However, it is evident from our studies³⁹ and others⁴⁵ that vascular NAD(P)H oxidase is regulated differently from phagocyte NAD(P)H oxidase, which may account for the difference in efficacy. Second, the higher concentration of cytosolic oxidase proteins and granule stores of flavocytochrome b also provides neutrophils with a ready supply of oxidase proteins to replenish inactive oxidase complexes.^28,46 Currently, there is no evidence for similar internal stores of NAD(P)H oxidase in vascular cells. Perhaps most importantly, degradation of peptides on the neutrophil surface by exoproteases is likely to prevent them from reaching the assembling NAD(P)H oxidase complex, necessitating the addition of higher peptide concentrations to achieve similar levels of inhibitory activity.^47,48 Finally, although a one amino acid difference in the gp91ds of human neutrophils could explain a lower efficacy of the mouse gp91ds, it is unlikely to explain such a marked reduction in activity. This difference in the efficacy of gp91ds-tat in the vasculature versus neutrophils may provide a therapeutic advantage, allowing inhibition of vascular O₂^- production while preserving normal neutrophil antimicrobial function.

We previously showed that infusion of Ang II into mice caused a significant increase in NAD(P)H oxidase components and O₂^-, mediated through the angiotensin type 1 (AT₁) receptor and accompanied by SBP elevation.¹² This was consistent with a previous report by Laursen et al¹¹ in rats showing that Ang II– but not norepinephrine-dependent hypertension increases vascular O₂^- production. In their model, liposome-encapsulated SOD was capable of partially attenuating the increase in blood pressure.¹¹ Likewise, our present data show a partial attenuation of Ang II–induced hypertension by gp91ds-tat. Since gp91ds-tat only partially attenuated the development of hypertension in the Ang II–treated mice, but completely reduced Ang II–increased O₂^- in the aorta, a partial contribution of O₂^- from NAD(P)H oxidase in the development of hypertension is suggested. A partial contribution by this enzyme or class of enzymes is not surprising, as the hypertensive effects of Ang II are complex,⁴⁹ and much of the increase in blood pressure is likely related to direct activation of AT₁ receptors, excitation/contraction,⁴⁹ and a variety of other central and peripheral effects.⁵⁰ Indeed, Laursen et al¹¹ showed that after 5 days of infusion, Ang II (0.75 mg · kg^-1 · d^-1) impaired aortic endothelium-dependent relaxation ex vivo, which was restored by cotreatment with liposomal SOD; however, it did not completely normalize blood pressure. Moreover, whereas NAD(P)H oxidase is a major source of O₂^- in the aorta,¹⁴ these studies have not been addressed in resistance vessels to our knowledge. It is plausible that the degree of O₂^- inhibition in resistance vessels will more accurately reflect the blood pressure–lowering effect of gp91ds-tat. These and other studies will be necessary to determine whether greater reductions in blood pressure might be achieved with higher concentrations of gp91ds-tat.

In summary, these data provide evidence for the involvement of gp91^phox or its homologues in vascular O₂^- production and for ongoing assembly with p47^phox, contributing to vascular O₂^- production and blood pressure elevation. Moreover, inhibition of this NAD(P)H oxidase is likely to promote vascular protection in a variety of disease states in which this enzyme is activated and implicated in vascular remodeling, such as restenosis and atherosclerosis.^51,52 This novel inhibitor could avoid compensatory and developmental issues associated with oxidase deletion and permit inhibition to be targeted to various cell types.

	Acknowledgments

 
This work was supported by NIH NHLBI Grants R01 HL55425, R01 AR42426, HL28982, and HL66575 and American Heart Association Grants 95011900 and 9808086W. We would like to thank Laura Nelson and Carl Polomski for technical assistance.

	Footnotes

 
Presented in part at the 72nd Scientific Sessions of the American Heart Association, Atlanta, Ga, November 7–10, 1999, and published in abstract form (Circulation. 1999;100[lsqb]suppl I[rsqb]:I-45) and at the Experimental Biology Meeting, San Diego, Calif, April 15–18, 2000, and published in abstract form (FASEB J. 2000;14:A119).

Received September 11, 2000; accepted July 13, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Omar HA, Cherry PD, Mortelliti MP, Burke-Wolin TM, Wolin MS. Inhibition of coronary artery superoxide dismutase attenuates endothelium-dependent and -independent nitrovasodilator relaxation. Circ Res. . 1991; 69: 601–608.[Abstract]

2. Rubanyi GM, Vanhoutte PM. Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. Am J Physiol. . 1986; 250: H822–H827.[Medline]

3. Mugge A, Elwell JH, Peterson TE, Harrison DG. Release of intact endothelium-derived relaxing factor depends on endothelial superoxide dismutase activity. Am J Physiol. . 1991; 260: C219–C225.[Medline]

4. Gryglewski RJ, Palmer RMJ, Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature. . 1986; 320: 454–456.[Medline]

5. Tesfamariam B, Cohen RA. Free radicals mediate endothelial cell dysfunction caused by elevated glucose. Am J Physiol. . 1992; 263: H321–H326.[Medline]

6. Katusic ZS, Vanhoutte PM. Superoxide anion is an endothelium-derived contracting factor. Am J Physiol. . 1989; 257: H33–H37.[Medline]

7. Wu L, de Champlain J. Effects of superoxide on signaling pathways in smooth muscle cells from rats. Hypertension. . 1999; 34: 1247–1253.[Abstract/Full Text]

8. Rajagopalan S, Kurz S, Münzel T, Tarpey M, Freeman BA, Griendling KK, Harrison DG. Angiotensin II -mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation: contribution to alterations of vasomotor tone. J Clin Invest. . 1996; 97: 1916–1923.[Abstract/Full Text]

9. Wang HD, Hope S, Du Y, Quinn MT, Cayatte A, Pagano PJ, Cohen RA. Paracrine role of adventitial superoxide anion in mediating spontaneous tone of the isolated rat aorta in angiotensin II–induced hypertension. Hypertension. . 1999; 33: 1225–1232.[Abstract/Full Text]

10. Wang HD, Pagano PJ, Du Y, Cayatte AJ, Quinn MT, Brecher P, Cohen RA. Superoxide anion from the adventitia of the rat thoracic aorta inactivates nitric oxide. Circ Res. . 1998; 82: 810–818.[Abstract/Full Text]

11. Laursen JB, Rajagopalan S, Galis Z, Tarpey M, Freeman BA, Harrison DG. Role of superoxide in angiotensin II–induced but not catecholamine-induced hypertension. Circulation. . 1997; 95: 588–593.[Abstract/Full Text]

12. Cifuentes ME, Rey FE, Carretero OA, Pagano PJ. Upregulation of p67^phox and gp91^phox in aortas from angiotensin II-infused mice. Am J Physiol Heart Circ Physiol. . 2000; 279: H2234–H2240.[Medline]

13. Mohazzab-H KM, Wolin MS. Sites of superoxide anion production detected by lucigenin in calf pulmonary artery smooth muscle. Am J Physiol. . 1994; 267: L815–L822.[Medline]

14. Pagano PJ, Ito Y, Tornheim K, Gallop PM, Tauber AI, Cohen RA. An NADPH oxidase superoxide-generating system in the rabbit aorta. Am J Physiol. . 1995; 268: H2274–H2280.[Medline]

15. Fukui T, Ishizaka N, Rajagopalan S, Laursen JB, Capers Q IV, Taylor WR, Harrison DG, de Leon H, Wilcox JN, Griendling KK. p22^phox mRNA expression and NADPH oxidase activity are increased in aortas from hypertensive rats. Circ Res. . 1997; 80: 45–51.[Abstract/Full Text]

16. Pagano PJ, Chanock SJ, Siwik DA, Colucci WS, Clark JK. Angiotensin II induces p67^phox mRNA expression and NADPH oxidase superoxide generation in rabbit aortic adventitial fibroblasts. Hypertension. . 1998; 32: 331–337.[Abstract/Full Text]

17. Kumar KV, Das UN. Are free radicals involved in the pathobiology of human essential hypertension? Free Radic Res Commun. . 1993; 19: 59–66.[Medline]

18. Bayraktutan U, Draper N, Lang D, Shah AM. Expression of functional neutrophil-type NADPH oxidase in cultured rat coronary microvascular endothelial cells. Cardiovasc Res. . 1998; 38: 256–262.[Medline]

19. Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res. . 1994; 74: 1141–1148.[Abstract]

20. Inanami O, Johnson JL, McAdara JK, El Benna J, Faust LP, Newburger PE, Babior BM. Activation of the leukocyte NADPH oxidase by phorbol ester requires the phosphorylation of p47^phox on serine 303 or 304. J Biol Chem. . 1998; 273: 9539–9543.[Abstract/Full Text]

21. Heitzer T, Wenzel U, Hink U, Krollner D, Skatchkov M, Stahl RAK, Macharzina R, Bräsen JH, Meinertz T, Münzel T. Increased NAD (P)H oxidase-mediated superoxide production in renovascular hypertension: evidence for an involvement of protein kinase C. Kidney Int. . 1999; 56: 252–260.

22. Meyer JW, Holland JA, Ziegler LM, Chang M-M, Beebe G, Schmitt ME. Identification of a functional leukocyte-type NADPH oxidase in human endothelial cells: a potential atherogenic source of reactive oxygen species. Endothelium. . 1999; 7: 11–22.[Medline]

23. Patterson C, Ruef J, Madamanchi NR, Barry-Lane P, Hu Z, Horaist C, Ballinger CA, Brasier AR, Bode C, Runge MS. Stimulation of a vascular smooth muscle cell NAD(P)H oxidase by thrombin: evidence that p47^phox may participate in forming this oxidase in vitro and in vivo. J Biol Chem. . 1999; 274: 19814–19822.[Abstract/Full Text]

24. Schieffer B, Luchtefeld M, Braun S, Hilfiker A, Hilfiker-Kleiner D, Drexler H. Role of NAD(P)H oxidase in angiotensin II-induced JAK/STAT signaling and cytokine induction. Circ Res. . 2000; 87: 1195–1201.[Abstract/Full Text]

25. Suh Y-A, Arnold RS, Lassegue B, Shi J, Xu X, Sorescu D, Chung AB, Griendling KK, Lambeth JD. Cell transformation by the superoxide-generating oxidase Mox1 [letter]. Nature. . 1999; 401: 79–82.[Medline]

26. Geiszt M, Kopp JB, Várnai P, Leto TL. Identification of Renox, an NAD(P)H oxidase in kidney. Proc Natl Acad Sci USA. . 2000; 97: 8010–8014.[Abstract/Full Text]

27. Lambeth JD, Cheng G, Arnold RS, Edens WA. Novel homologs of gp91^phox. Trends Biochem Sci. . 2000; 25: 459–461.[Medline]

28. DeLeo FR, Quinn MT. Assembly of the phagocyte NADPH oxidase: molecular interaction of oxidase proteins. J Leukoc Biol. . 1996; 60: 677–691.[Abstract]

29. DeLeo FR, Yu L, Burritt JB, Loetterle LR, Bond CW, Jesaitis AJ, Quinn MT. Mapping sites of interaction of p47^phox and flavocytochrome b with random-sequence peptide phage display libraries. Proc Natl Acad Sci USA. . 1995; 92: 7110–7114.[Abstract]

30. Bjorgvinsdottir H, Zhen L, Dinauer MC. Cloning of murine gp91^phox cDNA and functional expression in a human X-linked chronic granulomatous disease cell line. Blood. . 1996; 87: 2005–2010.[Abstract]

31. Lassègue B, Sorescu D, Szöcs K, Yin Q, Akers M, Zhang Y, Grant SL, Lambeth JD, Griendling KK. Novel gp91^phox homologues in vascular smooth muscle cells: nox1 mediates angiotensin II–induced superoxide formation and redox-sensitive signaling pathways. Circ Res. . 2001; 88: 888–894.[Abstract/Full Text]

32. Fawell S, Seery J, Daikh Y, Moore C, Chen LL, Pepinsky B, Barsoum J. Tat-mediated delivery of heterologous proteins into cells. Proc Natl Acad Sci USA. . 1994; 91: 664–668.[Abstract]

33. Kim DT, Mitchell DJ, Brockstedt DG, Fong L, Nolan GP, Fathman CG, Engleman EG, Rothbard JB. Introduction of soluble proteins into the MHC class I pathway by conjugation to an HIV tat peptide. J Immunol. . 1997; 159: 1666–1668.[Abstract]

34. Miller FJ Jr, Gutterman DD, Rios CD, Heistad DD, Davidson BL. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res. . 1998; 82: 1298–1305.[Abstract/Full Text]

35. DeLeo FR, Jutila MA, Quinn MT. Characterization of peptide diffusion into electropermeabilized neutrophils. J Immunol Methods. . 1996; 198: 35–49.[Medline]

36. Lokesh BR, Cunningham ML. Further studies on the formation of oxygen radicals by potassium superoxide in aqueous medium for biochemical investigations. Toxicol Lett. . 1986; 34: 75–84.[Medline]

37. Doussiere J, Vignais PV. Diphenylene iodonium as an inhibitor of the NADPH oxidase complex of bovine neutrophils. Eur J Biochem. . 1992; 208: 61–71.[Abstract]

38. Stuehr DJ, Fasehun OA, Kwon NS, Gross SS, Gonzalez JA, Levi R, Nathan CF. Inhibition of macrophage and endothelial cell nitric oxide synthase by diphenyleneiodonium and its analogs. FASEB J. . 1991; 5: 98–103.[Abstract]

39. Pagano PJ, Clark JK, Cifuentes-Pagano ME, Clark SM, Callis GM, Quinn MT. Localization of a constitutively active, phagocyte-like NADPH oxidase in rabbit aortic adventitia: enhancement by angiotensin II. Proc Natl Acad Sci USA. . 1997; 94: 14483–14488.[Abstract/Full Text]

40. Jones SA, O’Donnell VB, Wood JD, Broughton JP, Hughes EJ, Jones OTG. Expression of phagocyte NADPH oxidase components in human endothelial cells. Am J Physiol. . 1996; 271: H1626–H1634.[Medline]

41. Hsich E, Segal BH, Pagano PJ, Rey FE, Paigen B, Deleonardis J, Hoyt RF, Holland SM, Finkel T. Vascular effects following homozygous disruption of p47^phox: an essential component of NADPH oxidase. Circulation. . 2000; 101: 1234–1236.[Medline]

42. Korthuis RJ, Gute DC, Blecha F, Ross CR. PR-39, a proline/arginine-rich antimicrobial peptide, prevents postischemic microvascular dysfunction. Am J Physiol. . 1999; 277 (3 pt 2)H1007–H1013.[Medline]

43. Stolk J, Hiltermann TJN, Dijkman JH, Verhoeven AJ. Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol. Am J Respir Cell Mol Biol. . 1994; 11: 95–102.[Abstract]

44. Klebanoff SJ. Myeloperoxidase. Proc Assoc Am Physicians. . 2001; 111: 383–389.

45. Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res. . 2000; 86: 494–501.[Abstract/Full Text]

46. Clark RA. Activation of the neutrophil respiratory burst oxidase. J Infect Dis. . 1999; 179: S309–S317.[Medline]

47. Shipp MA, Stefano GB, Switzer SN, Griffin JD, Reinherz EL. CD10 (CALLA)/neutral endopeptidase 24.11 modulates inflammatory peptide-induced changes in neutrophil morphology, migration, and adhesion proteins and is itself regulated by neutrophil activation. Blood. . 1991; 78: 1834–1841.[Abstract]

48. Owen CA, Campbell MA, Sannes PL, Boukedes SS, Campbell EJ. Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteases. J Cell Biol. . 1995; 131: 775–789.[Abstract]

49. Kim S, Iwao H. Molecular and cellular mechanisms of angiotensin II-mediated cardiovascular and renal diseases. Pharmacol Rev. . 2000; 52: 11–34.[Abstract/Full Text]

50. Simon G, Abraham G. Angiotensin II administration as an experimental model of hypertension. In: Laragh JH, Brenner BM, eds. Hypertension: Pathophysiology, Diagnosis, and Management. New York, NY: Raven Press; 1995: 1423–1435.

51. Azevedo LCP, Pedro MDA, Souza LC, de Souza HP, Janiszewski M, da Luz PL, Laurindo FRM. Oxidative stress as a signaling mechanism of the vascular response to injury: the redox hypothesis of restenosis. Cardiovasc Res. . 2000; 47: 436–445.[Medline]

52. Warnholtz A, Nickenig G, Schulz E, Macharzina R, Bräsen JH, Skatchkov M, Heitzer T, Stasch JP, Griendling KK, Harrison DG, Böhm M, Meinertz T, Münzel T. Increased NADH -oxidase-mediated superoxide production in the early stages of atherosclerosis: evidence for involvement of the renin-angiotensin system. Circulation. . 1999; 99: 2027–2033.[Abstract/Full Text]

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				L. Park, J. Anrather, P. Zhou, K. Frys, R. Pitstick, S. Younkin, G. A. Carlson, and C. Iadecola NADPH Oxidase-Derived Reactive Oxygen Species Mediate the Cerebrovascular Dysfunction Induced by the Amyloid {beta} Peptide J. Neurosci., February 16, 2005; 25(7): 1769 - 1777. [Abstract] [Full Text] [PDF]

				H. M. Dourron, G. M. Jacobson, J. L. Park, J. Liu, D. J. Reddy, M. L. Scheel, and P. J. Pagano Perivascular gene transfer of NADPH oxidase inhibitor suppresses angioplasty-induced neointimal proliferation of rat carotid artery Am J Physiol Heart Circ Physiol, February 1, 2005; 288(2): H946 - H953. [Abstract] [Full Text] [PDF]

				R. Furst, C. Brueckl, W. M. Kuebler, S. Zahler, F. Krotz, A. Gorlach, A. M. Vollmar, and A. K. Kiemer Atrial Natriuretic Peptide Induces Mitogen-Activated Protein Kinase Phosphatase-1 in Human Endothelial Cells via Rac1 and NAD(P)H Oxidase/Nox2-Activation Circ. Res., January 7, 2005; 96(1): 43 - 53. [Abstract] [Full Text] [PDF]

				N. R. Madamanchi, A. Vendrov, and M. S. Runge Oxidative Stress and Vascular Disease Arterioscler Thromb Vasc Biol, January 1, 2005; 25(1): 29 - 38. [Abstract] [Full Text] [PDF]

				N. Lu, B. G. Helwig, R. J. Fels, S. Parimi, and M. J. Kenney Central Tempol alters basal sympathetic nerve discharge and attenuates sympathetic excitation to central ANG II Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2626 - H2633. [Abstract] [Full Text] [PDF]

				K. Kazama, J. Anrather, P. Zhou, H. Girouard, K. Frys, T. A. Milner, and C. Iadecola Angiotensin II Impairs Neurovascular Coupling in Neocortex Through NADPH Oxidase-Derived Radicals Circ. Res., November 12, 2004; 95(10): 1019 - 1026. [Abstract] [Full Text] [PDF]

				J.-M. Li and A. M Shah Endothelial cell superoxide generation: regulation and relevance for cardiovascular pathophysiology Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2004; 287(5): R1014 - R1030. [Abstract] [Full Text] [PDF]

				S. Adler and H. Huang Oxidant stress in kidneys of spontaneously hypertensive rats involves both oxidase overexpression and loss of extracellular superoxide dismutase Am J Physiol Renal Physiol, November 1, 2004; 287(5): F907 - F913. [Abstract] [Full Text] [PDF]

				M. T. Quinn and K. A. Gauss Structure and regulation of the neutrophil respiratory burst oxidase: comparison with nonphagocyte oxidases J. Leukoc. Biol., October 1, 2004; 76(4): 760 - 781. [Abstract] [Full Text] [PDF]

				L. Park, J. Anrather, P. Zhou, K. Frys, G. Wang, and C. Iadecola Exogenous NADPH Increases Cerebral Blood Flow Through NADPH Oxidase-Dependent and -Independent Mechanisms Arterioscler Thromb Vasc Biol, October 1, 2004; 24(10): 1860 - 1865. [Abstract] [Full Text] [PDF]

				J. Liu, A. Ormsby, N. Oja-Tebbe, and P. J. Pagano Gene Transfer of NAD(P)H Oxidase Inhibitor to the Vascular Adventitia Attenuates Medial Smooth Muscle Hypertrophy Circ. Res., September 17, 2004; 95(6): 587 - 594. [Abstract] [Full Text] [PDF]

				G. Wang, J. Anrather, J. Huang, R. C. Speth, V. M. Pickel, and C. Iadecola NADPH Oxidase Contributes to Angiotensin II Signaling in the Nucleus Tractus Solitarius J. Neurosci., June 16, 2004; 24(24): 5516 - 5524. [Abstract] [Full Text] [PDF]

				M. J. Ryan, S. P. Didion, S. Mathur, F. M. Faraci, and C. D. Sigmund Angiotensin II-Induced Vascular Dysfunction Is Mediated by the AT1A Receptor in Mice Hypertension, May 1, 2004; 43(5): 1074 - 1079. [Abstract] [Full Text] [PDF]

				O. Jung, J.G. Schreiber, H. Geiger, T. Pedrazzini, R. Busse, and R.P. Brandes gp91phox-Containing NADPH Oxidase Mediates Endothelial Dysfunction in Renovascular Hypertension Circulation, April 13, 2004; 109(14): 1795 - 1801. [Abstract] [Full Text] [PDF]

				J.-M. Li, S. Wheatcroft, L. M. Fan, M. T. Kearney, and A. M. Shah Opposing Roles of p47phox in Basal Versus Angiotensin II-Stimulated Alterations in Vascular O2- Production, Vascular Tone, and Mitogen-Activated Protein Kinase Activation Circulation, March 16, 2004; 109(10): 1307 - 1313. [Abstract] [Full Text] [PDF]

				J. Ou, J. T. Fontana, Z. Ou, D. W. Jones, A. W. Ackerman, K. T. Oldham, J. Yu, W. C. Sessa, and K. A. Pritchard Jr. Heat shock protein 90 and tyrosine kinase regulate eNOS NO{middle dot} generation but not NO{middle dot} bioactivity Am J Physiol Heart Circ Physiol, February 1, 2004; 286(2): H561 - H569. [Abstract] [Full Text] [PDF]

				M. Z. Haque and D. S. A. Majid Assessment of Renal Functional Phenotype in Mice Lacking gp91PHOX Subunit of NAD(P)H Oxidase Hypertension, February 1, 2004; 43(2): 335 - 340. [Abstract] [Full Text] [PDF]

				E. Werner GTPases and reactive oxygen species: switches for killing and signaling J. Cell Sci., January 15, 2004; 117(2): 143 - 153. [Abstract] [Full Text] [PDF]

				T. Munzel, R. Feil, A. Mulsch, S. M. Lohmann, F. Hofmann, and U. Walter Physiology and Pathophysiology of Vascular Signaling Controlled by Cyclic Guanosine 3',5'-Cyclic Monophosphate-Dependent Protein Kinase Circulation, November 4, 2003; 108(18): 2172 - 2183. [Full Text] [PDF]

				S. Fujii, L. Zhang, J. Igarashi, and H. Kosaka L-Arginine Reverses p47phox and gp91phox Expression Induced by High Salt in Dahl Rats Hypertension, November 1, 2003; 42(5): 1014 - 1020. [Abstract] [Full Text] [PDF]

				K. K. Griendling and G. A. FitzGerald Oxidative Stress and Cardiovascular Injury: Part II: Animal and Human Studies Circulation, October 28, 2003; 108(17): 2034 - 2040. [Full Text] [PDF]

				O. Jung, S. L. Marklund, H. Geiger, T. Pedrazzini, R. Busse, and R. P. Brandes Extracellular Superoxide Dismutase Is a Major Determinant of Nitric Oxide Bioavailability: In Vivo and Ex Vivo Evidence From ecSOD-Deficient Mice Circ. Res., October 3, 2003; 93(7): 622 - 629. [Abstract] [Full Text] [PDF]

				D. Gregg, F. M. Rauscher, and P. J. Goldschmidt-Clermont Rac regulates cardiovascular superoxide through diverse molecular interactions: more than a binary GTP switch Am J Physiol Cell Physiol, October 1, 2003; 285(4): C723 - C734. [Abstract] [Full Text] [PDF]

				N. Lopes, D. Gregg, S. Vasudevan, H. Hassanain, P. Goldschmidt-Clermont, and H. Kovacic Thrombospondin 2 Regulates Cell Proliferation Induced by Rac1 Redox-Dependent Signaling Mol. Cell. Biol., August 1, 2003; 23(15): 5401 - 5408. [Abstract] [Full Text] [PDF]

				B. Lassegue and R. E. Clempus Vascular NAD(P)H oxidases: specific features, expression, and regulation Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2003; 285(2): R277 - R297. [Abstract] [Full Text] [PDF]

				H.-Y. Sohn, F. Krotz, T. Gloe, M. Keller, K. Theisen, V. Klauss, and U. Pohl Differential regulation of xanthine and NAD(P)H oxidase by hypoxia in human umbilical vein endothelial cells. Role of nitric oxide and adenosine Cardiovasc Res, June 1, 2003; 58(3): 638 - 646. [Abstract] [Full Text] [PDF]

				J. Liu, F. Yang, X.-P. Yang, M. Jankowski, and P. J. Pagano NAD(P)H Oxidase Mediates Angiotensin II-Induced Vascular Macrophage Infiltration and Medial Hypertrophy Arterioscler Thromb Vasc Biol, May 1, 2003; 23(5): 776 - 782. [Abstract] [Full Text] [PDF]

				R. P. Brandes A Radical Adventure: The Quest for Specific Functions and Inhibitors of Vascular NAPDH Oxidases Circ. Res., April 4, 2003; 92(6): 583 - 585. [Full Text] [PDF]

				G. M. Jacobson, H. M. Dourron, J. Liu, O. A. Carretero, D. J. Reddy, T. Andrzejewski, and P. J. Pagano Novel NAD(P)H Oxidase Inhibitor Suppresses Angioplasty-Induced Superoxide and Neointimal Hyperplasia of Rat Carotid Artery Circ. Res., April 4, 2003; 92(6): 637 - 643. [Abstract] [Full Text] [PDF]

				J.-M. Li and A. M. Shah Mechanism of Endothelial Cell NADPH Oxidase Activation by Angiotensin II. ROLE OF THE p47phox SUBUNIT J. Biol. Chem., March 28, 2003; 278(14): 12094 - 12100. [Abstract] [Full Text] [PDF]

				P. T. Schumacker Angiotensin II Signaling in the Brain: Compartmentalization of Redox Signaling? Circ. Res., November 29, 2002; 91(11): 982 - 984. [Full Text] [PDF]

				F. E. Rey, X.-C. Li, O. A. Carretero, J. L. Garvin, and P. J. Pagano Perivascular Superoxide Anion Contributes to Impairment of Endothelium-Dependent Relaxation: Role of gp91phox Circulation, November 5, 2002; 106(19): 2497 - 2502. [Abstract] [Full Text] [PDF]

				U. Landmesser, H. Cai, S. Dikalov, L. McCann, J. Hwang, H. Jo, S. M. Holland, and D. G. Harrison Role of p47phox in Vascular Oxidative Stress and Hypertension Caused by Angiotensin II Hypertension, October 1, 2002; 40(4): 511 - 515. [Abstract] [Full Text] [PDF]

				F. Krotz, H. Y. Sohn, T. Gloe, S. Zahler, T. Riexinger, T. M. Schiele, B. F. Becker, K. Theisen, V. Klauss, and U. Pohl NAD(P)H oxidase-dependent platelet superoxide anion release increases platelet recruitment Blood, July 18, 2002; 100(3): 917 - 924. [Abstract] [Full Text] [PDF]

				A. Csiszar, Z. Ungvari, J. G. Edwards, P. Kaminski, M. S. Wolin, A. Koller, and G. Kaley Aging-Induced Phenotypic Changes and Oxidative Stress Impair Coronary Arteriolar Function Circ. Res., June 14, 2002; 90(11): 1159 - 1166. [Abstract] [Full Text] [PDF]

				R. M. Touyz, X. Chen, F. Tabet, G. Yao, G. He, M. T. Quinn, P. J. Pagano, and E. L. Schiffrin Expression of a Functionally Active gp91phox-Containing Neutrophil-Type NAD(P)H Oxidase in Smooth Muscle Cells From Human Resistance Arteries: Regulation by Angiotensin II Circ. Res., June 14, 2002; 90(11): 1205 - 1213. [Abstract] [Full Text] [PDF]

				B. Lassegue and K. K. Griendling Out Phoxing the Endothelium: What's Left Without p47? Circ. Res., February 8, 2002; 90(2): 123 - 124. [Full Text] [PDF]

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