Published online before print August 16, 2001, doi: 10.1161/hh1701.096037
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© 2001 American Heart Association, Inc.
Integrative Physiology |
Novel Competitive Inhibitor of NAD(P)H Oxidase Assembly Attenuates Vascular O2- and Systolic Blood Pressure in Mice
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
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Abstract |
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We previously reported enhanced expression of the p67phox and gp91phox 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 p47phox association with gp91phox 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 gp91phox (known to interact with p47phox). 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 p47phox and gp91phox (or its homologues) can block O2- production and attenuate blood pressure elevation in mice.
Key Words: superoxide • angiotensin II • NAD(P)H oxidase • gp91phox • p47phox
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Introduction |
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Superoxide anion (O2-) can interfere with nitric oxide (NO)–dependent vasodilatation1–4 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 p22phox and p67phox15,16 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 p47phox, which then binds to membrane-associated gp91phox.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, p47phox 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 gp91phox, its smooth muscle homologue nox1-containing NAD(P)H oxidase,25 or nox426,27 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 gp91phox, which are involved in the binding of gp91phox to p47phox, 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 gp91phox 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 p47phox 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 p47phox translocation to plasma membranes in smooth muscle cells,24 which have been shown to contain primarily nox1 and nox4,31 interaction of p47phox with anchoring gp91phox-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 gp91phox 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-.
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Materials and Methods |
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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.
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Results |
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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).
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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).
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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).
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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).
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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.
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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).
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Discussion |
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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 gp91phox in aortas of rabbits14,39 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 gp91phox at the N-terminal end, which contains regions that interact with p47phox. Based on this knowledge, we developed a competitive antagonist of the interaction of gp91phox and p47phox 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, gp91phox, p47phox, p67phox, and p22phox, in the vascular wall22,39,40 and observed upregulation of p22phox, gp91phox, and p67phox by Ang II, concomitant with significant elevations in O2-.12,15 In addition, p47phox 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 gp91phox and p47phox 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 p47phox is activated in neutrophils, it is phosphorylated, which enhances its interaction with p67phox. This complex then translocates to the inner surface of the plasma membrane where p47phox interacts with gp91phox, 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 gp91phox and p47phox.
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 gp91phox12 and nox1, a shorter homologue of gp91phox 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 (gp91phox and nox1). Future studies will be necessary to directly examine the ability of this peptide to inhibit assembly of nox1 and nox4 with p47phox.
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 gp91phox or its homologues in vascular O2- production and for ongoing assembly with p47phox, 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.
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Acknowledgments |
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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.
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Footnotes |
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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.
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 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]
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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]
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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]
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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]
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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]
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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]
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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]
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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]
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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]
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 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]
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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]
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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]
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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]
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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]
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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]
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 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]
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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]
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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]
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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]
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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]
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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]
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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]
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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]
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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]
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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]
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 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]
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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]
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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]
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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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