(Circulation. 2001;104:79.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Genetic Demonstration of p47phox-Dependent Superoxide Anion Production in Murine Vascular Smooth Muscle Cells
From the Laboratory of Host Defenses, National Institutes of Health, National Institute of Allergy and Infectious Diseases, Bethesda, Md.
Correspondence to Dr Thomas L. Leto, National Institutes of Health, Building 10, Room 11N106, 10 Center Dr, Bethesda, MD 20892. E-mail tleto{at}nih.gov
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Abstract |
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Background—Previous investigations provide evidence that an enzyme related to the phagocyte NADPH oxidase produces superoxide in the blood vessel wall. These data, however, are confounded by observations that both NADPH and NADH serve as substrates for superoxide production in vascular cells. To clarify this issue, we compared the superoxide-generating capabilities of vascular smooth muscle cells (VSMCs) derived from wild-type (p47phox+/+; phagocyte oxidase) mice with those from mice that lack p47phox (p47phox-/-; "knockout"), an essential component of the phagocyte NADPH oxidase.
Methods and Results—VSMCs were derived from aortic explants harvested from p47phox+/+ or p47phox-/- mice. VSMCs from p47phox+/+ but not those from p47phox-/- mice produced superoxide after stimulation by phorbol myristate acetate. Consistent with this, p47phox was detected only in p47phox+/+ VSMCs. p47phox-transduced p47phox-/- but not enhanced green fluorescent protein–transduced p47phox-/- VSMCs generated significant levels of superoxide after stimulation by angiotensin II or platelet-derived growth factor-BB (PDGF-BB). Enhanced expression of recombinant p47phox in p47phox-transduced p47phox-/- cells correlated with superoxide production in these cells.
Conclusions—These data provide direct functional proof that an oxidase requiring the p47phox component mediates superoxide release from VSMCs in the blood vessel wall in response to angiotensin II or PDGF-BB.
Key Words: muscle, smooth • NADPH oxidase • p47phox • transduction • superoxide
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In the cardiovascular system, multiple functions for reactive oxygen species (ROS) are becoming apparent, including signal transduction in vascular smooth muscle cells (VSMCs)1 and blood pressure homeostasis.2 A primary source of ROS, superoxide anion can be converted into hydrogen peroxide, which may be detrimental to cardiovascular performance through its ability to promote VSMC proliferation.3 Therefore, local vascular levels of superoxide anions may determine whether physiological or pathological circumstances prevail, which suggests that regulation of the production of these mediators may provide a novel therapeutic approach to intervention in cardiovascular disease.
Pharmacological or genetic control of ROS release in cardiovascular tissue necessitates the identification of the enzyme(s) responsible for their production. Several investigations have suggested that a variety of enzymes, including a phagocyte-like NADPH oxidase, function in the blood vessel wall. The phagocytic enzyme is composed of 5 essential components, including 2 that are membrane-bound: p22phox (phagocyte oxidase) and gp91phox, and 3 that are located in the cytosol: p47phox, p67phox, and Rac1 (guinea pig) or Rac2 (human).4 NADPH-dependent superoxide production detected in cell-free assays of nonstimulated rabbit aorta preparations was inhibited by diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase and other flavoenzymes,5 6 7 whereas NADH yielded less superoxide when used as a substrate.8 In contrast, NADH-dependent superoxide generation exhibited by human umbilical vein endothelial cell9 or rabbit aortic adventitial fibroblast preparations10 was greater than the activity observed when NADPH was used as a substrate. In both studies, basal NADPH-dependent superoxide production was preferentially inhibited by DPI compared with basal NADH-dependent activity. In contrast, angiotensin II (Ang II)–stimulated superoxide generation in adventitial fibroblasts was DPI-sensitive when either substrate was provided.10 Both immunochemical10 11 12 13 14 and genetic9 11 12 13 15 evidence suggest that phagocyte NADPH oxidase (phox) components are present and functional in blood vessels10 11 13 14 and vascular cells (endothelial cells, smooth muscle cells, and fibroblasts),9 10 11 12 13 15 although the substrate preferences and pharmacological sensitivities of the putative oxidases involving these components were not always explored. Gorlach et al11 recently demonstrated that gp91phox is involved in ROS production by endothelial cells by comparing aortic vessel segments derived from wild-type and gp91phox-deficient mice, although NADPH-dependent superoxide release in these whole tissues did not involve gp91phox. Together, the results of these investigations imply that several superoxide-producing enzymes function in the blood vessel wall, including more than one 1 enzyme that preferentially uses NADPH or NADH under basal conditions, and agonist-stimulated oxidases that are DPI-sensitive and use either substrate as a source of electrons.
In light of recent data that indicate the presence of p47phox in VSMCs and suggest a role for p47phox in VSMC mitogenesis,13 the present study was undertaken to address directly whether an oxidase requiring the p47phox component participates in agonist-stimulated superoxide production in VSMCs. By comparing the abilities of phorbol myristate acetate (PMA), Ang II, and platelet-derived growth factor (PDGF) to stimulate superoxide production in VSMCs derived from p47phox+/+ and p47phox-/- mice, our studies indicate that p47phox is an essential component of a superoxide-generating system in these cells.
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Methods |
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Derivation of Murine VSMCs
The p47phox-/- mouse, which was created as a murine model for chronic granulomatous disease by targeted disruption of the p47phox gene, has been described previously.16 For each set of experiments, aortas from 3 adult (12 to 16 weeks old) female C57BL/6 p47phox+/+ or C57BL/6 p47phox-/- mice were harvested 2 to 3 minutes after their death by cervical dislocation. The vessels were transferred into culture medium (SmGM-2; includes 5% FBS; Clonetics). The vessels were stripped of connective tissue, cut into rings (3 to 5 mm long), and placed onto sterile plastic 24-well or 6-well tissue culture plates. After
21 days, the cells were harvested for
experimentation.
Experimental Protocols
VSMCs derived from aortic explants of
p47phox+/+ or
p47phox-/- mice were collected and
distributed as follows: (1) for phase-contrast microscopy or indirect
immunofluorescence staining to detect a VSMC
antigenic marker, 
-smooth muscle
actin,17 or p47phox; (2) for
chemiluminescence measurement of superoxide production; or (3)
for serial passage and measurement of superoxide production as
follows: 10% of the population of cells of each genotype was
seeded into T-25-cm2 plastic
tissue-culture–treated flasks (passage 1, P1; Nalge NUNC
International). On reaching 90% to 100% confluence, the VSMCs were
trypsinized and distributed for chemiluminescence assays or propagation
as for P1 (ie, 10% of the P1 cells was distributed to a
T-25-cm2 flask; P2). Therefore, the relative
expansion of cultures was the product of the dilution factor for
each passage (10) multiplied by the relative density of cultures at
each passage.
Phase-Contrast and Indirect
Immunofluorescence Microscopy
Before harvest, cells derived from
p47phox+/+ or
p47phox-/- explants were observed by
phase-contrast microscopy and photographed. For this purpose, the cells
were rinsed twice with 1x PBS and left submerged in this medium for
microscopic viewing (Zeiss) and
photography.
VSMCs derived from p47phox+/+ and
p47phox-/- mice were harvested for
immunofluorescence detection of -smooth muscle
actin and p47phox. For the detection of -smooth muscle actin, a
monoclonal anti–-smooth muscle actin antibody (Clone 1A4;
Sigma) or the isotype control, mouse IgG2a
(Sigma) were used. These primary antibodies were detected with a Texas
red–conjugated sheep anti-mouse antibody
(Jackson ImmunoResearch). Detection of p47phox
was performed with specific goat polyclonal anti-human p47phox serum as
described.18
Measurement of ROS
To measure superoxide generated by VSMCs derived from
p47phox+/+ and
p47phox-/- mice, a mixture of a
superoxide anion–specific chemiluminescence indicator reagent
(Diogenes; National Diagnostics;
50% of total reaction volume) containing dimethylsulfoxide (vehicle
for PMA), human Ang II (10 nmol/L; Sigma), human PDGF-BB (5 ng/mL;
Sigma), or PMA (2 µg/mL; Sigma) was added to adherent VSMCs
previously cultured (1x104 cells/well,
37°C overnight) in sterile microtiter chemiluminescence plates (Dynex
Technologies). The cells were cultured in culture medium
(Figure 2
and
Table 1) or in medium consisting of SmBM (Clonetics)
containing 0.5% FBS, 1% penicillin-streptomycin, and 1% glutamine
(Figure 5 and
Table 2). The dose of each
physiological agonist used was determined on the
basis of earlier
investigations.10 19 20
Total superoxide dismutase (SOD)–inhibitable superoxide generation was
measured in a luminometer (Labsystems Luminoskan; 0.5-second readings
obtained at 1-minute intervals over 40 minutes).
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Retroviral Transduction of Murine VSMCs
VSMCs derived from
p47phox+/+ or
p47phox-/- mice were seeded into
T-75-cm2 flasks (Nalge NUNC International).
After reaching 75% confluence, cells were transduced for 3 consecutive
days with amphotropic packaged MFGS retrovirus by methods described
elsewhere.21 Briefly, the
cells were covered with medium containing enhanced green
fluorescent protein (EGFP; control) or human p47phox
cDNA21 in X-VIVO 10
(BioWhittaker) medium (diluted 1:1 with culture medium) including
protamine (6 µg/mL). These cultures were centrifuged at
525g for 20 minutes at 32°C
and incubated at 37°C for 8 hours, after which time the viral
supernatants were replaced with fresh culture medium. After overnight
incubation of the cells at 37°C, this spin-transduction protocol was
repeated for 2 consecutive days. These transduced cells were then
analyzed for p47phox production and superoxide
production 4 days after the initial
transduction.
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Results |
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Phase-Contrast Microscopy and Indirect Immunofluorescence Staining of
-Smooth
Muscle ActinFigure 1
depicts cells emerging from aortic explants
derived from p47phox+/+ (A) and
p47phox-/- (B) mice. Cells in both
cultures (p47phox+/+, C, and
p47phox-/-, D) assumed spindle shapes,
characteristic of VSMCs or adventitial fibroblasts. Indirect
immunofluorescence detection of -smooth muscle
actin (p47phox+/+, E, and
p47phox-/-, F), an antigenic marker of
VSMCs17 and an indicator of
VSMC phenotypic status,22
revealed no differences in the content (>95%) of positively stained
cells between cultures. These results confirmed the identity of these
cultures and indicated that a deficiency of p47phox did not produce
discernible morphological or phenotypic differences between
cells.
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Superoxide Production by Murine
VSMCs
To directly test whether p47phox is involved in
superoxide release by VSMCs, we compared the abilities of cultures
derived from p47phox+/+ and
p47phox-/- aortic explants to generate
superoxide. We considered the possibility that these cultures contained
tissue-derived macrophages, which could represent a
source of superoxide but would fail to divide in culture. To test this
possibility, we compared superoxide production between culture
passages against the extent of cell culture expansion between passages
(Figure 2
and
Table 1). To stimulate the cells, we used PMA (2 µg/mL),
a potent nonphysiological agonist used to
activate NADPH
oxidase.4
Figure 2 shows that basal production of superoxide
in P1 p47phox+/+ cultures was approximately
equivalent to that produced by P1
p47phox-/- cells. This low level of
superoxide release yielded by p47phox-/-
cells was not inhibited by DPI (8 µmol/L; not shown). Therefore,
nonstimulated superoxide release detected in these cultures could
originate from nonflavoenzymatic sources, such as
cyclooxygenases. PMA stimulated significant
superoxide production by P1
p47phox+/+ cells, which occurred within a
40-minute assay, but not by P1
p47phox-/- cells
(Figure 2). With each passage, superoxide production
declined, but not to the extent predicted for nondividing cells when
considering the extent of culture expansion
(Figure 2 and
Table 1). These observations suggest that superoxide
production in p47phox+/+ cultures
was derived from VSMCs, but not from tissue macrophages. In
addition, inspections of PMA-stimulated
p47phox+/+ culture stained with nitro blue
tetrazolium revealed no contaminating phagocytic cells with a high
superoxide output (not shown). We suspected that reductions in
superoxide release were related to reduced expression of NADPH oxidase
components during culture, analogous to that previously observed in
monocyte-derived macrophages maintained in
vitro.23
These observations appear to be inconsistent with those in a previous study, which indicated that PMA fails to stimulate ROS release from human VSMCs or endothelium-denuded aortic segments (rat and mouse)11 ; other authors, however, reported that PMA stimulates ROS production in VSMCs.24 Differences in PMA doses or sensitivities of the superoxide-detecting systems may account for these discrepancies.
Endogenous p47phox was selectively detected in
P1 p47phox+/+ VSMCs
(Figure 3A; corresponding phase-contrast image in
Figure 3C), indicating that the p47phox deficiency in
p47phox-/- VSMCs
(Figure 3B; corresponding phase-contrast image in
Figure 3D) may account for their inability to produce
superoxide after PMA stimulation.
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Superoxide Production in
p47phox-/- VSMCs Transduced With
Recombinant p47phox
Together, the results presented in
Figures 2 and 3 suggest that the ability of
p47phox+/+ VSMCs to produce superoxide is
related to selective expression of p47phox in these cells. To test this
hypothesis, P1, 3-week-old p47phox+/+ and
p47phox-/- VSMC cultures were
retrovirally transduced with human recombinant p47phox, and superoxide
production was measured in response to stimulation by Ang II or
PDGF-BB. These agonists are relevant because they induce reactive
oxidant release by vascular
cells10 13 15 19 20 25 26
and seem to play roles in the pathogeneses of
hypertension2 and
atherosclerosis.27
Figure 4 demonstrates significant fluorescence
staining in EGFP-transduced (control)
p47phox+/+ (A) and
p47phox-/- (B) VSMCs and in
p47phox-transduced p47phox+/+ (C) and
p47phox-/- (D) VSMCs. Microscopic
inspections indicated that the efficiency of transduction by each virus
was 90% to 100%. The extent of immunofluorescence
staining in p47phox-transduced VSMCs indicated abundant expression of
recombinant p47phox in these cells relative to untransduced cells
(Figure 3A and 3B).
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In the absence of stimulation, the amount of superoxide
released by EGFP-transduced and p47phox-transduced cells was not
significantly different
(Table 2). Under agonist-stimulated conditions, superoxide
production correlated with p47phox expression in
p47phox-transduced p47phox-/- VSMCs in
response to Ang II (10 nmol/L;
Figure 5A and
Table 2) or PDGF-BB (5 ng/mL;
Figure 5B and
Table 2). In contrast, EGFP-transduced
p47phox-/- VSMCs did not produce
superoxide after stimulation by either agonist
(Figure 5A and 5B and
Table 2), indicating that retroviral transduction itself
was insufficient to restore agonist-induced superoxide generation.
Superoxide production yielded by p47phox-transduced
p47phox-/- VSMCs in response to Ang II
or PDGF-BB was comparable to that by EGFP-transduced
p47phox+/+ or p47phox-transduced
p47phox+/+ VSMCs
(Figure 5A and 5B and
Table 2). Furthermore, agonist-stimulated superoxide
release by EGFP-transduced p47phox+/+ and
p47phox-transduced p47phox-/- and
p47phox+/+ VSMCs was significantly greater
than that yielded under nonstimulated conditions. The levels of
superoxide produced by VSMCs after stimulation by either agonist,
however, were significantly lower than those yielded by PMA stimulation
(Table 1, 3 weeks after harvest of aortas). Our data
provide direct evidence that p47phox plays a role in superoxide
production by VSMCs in response to
physiological agonists.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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We provide genetic proof that p47phox participates in superoxide production in murine VSMCs. The selective ability of p47phox+/+ VSMCs to produce superoxide was not attributed to phenotypic differences between p47phox+/+ and p47phox-/- cultures, on the basis of indistinguishable
-smooth muscle actin staining patterns. Instead,
superoxide production in p47phox+/+
VSMCs was attributed to the exclusive expression of p47phox, because
transduction of p47phox-/- VSMCs with
recombinant human p47phox cDNA restored agonist-induced superoxide
production. Ang II– or PDGF-stimulated superoxide
production by EGFP-transduced
p47phox+/+, p47phox-transduced
p47phox-/-, and p47phox-transduced
p47phox+/+ VSMCs was significantly greater
than that by nonstimulated cells. Our results provide direct functional evidence that a phagocyte-like NADPH oxidase involving p47phox produces superoxide in VSMCs. In phagocytes, p47phox and other cytosolic proteins are essential cofactors of the enzyme, which do not themselves produce superoxide.4 Previous demonstrations of p22phox-dependent15 and Rac1-dependent28 superoxide production in VSMCs provide further evidence that a phagocyte-like oxidase functions in VSMCs. Interestingly, p22phox is reportedly not stabilized in the absence of gp91phox in phagocytes29 ; gp91phox, however, was not detected in VSMCs.11 15 A homologue of gp91phox, such as Mox1, may act as the core electron transfer component of the VSMC oxidase.11 30 p67phox was not detected in VSMCs13 but appears to participate in superoxide production in rabbit aortic adventitial fibroblasts.10 The disparities in oxidase compositions between phagocytes and VSMCs may explain differences in their primary physiological roles and their distinct responses to different agonists. Indeed, superoxide release from VSMCs and phagocytes seems to have preferential roles in signal transduction1 and host defense,4 respectively. Further investigations are necessary to delineate the partners of p47phox that facilitate superoxide release by VSMCs or establish whether Mox1 could fulfil such a role.
In the blood vessel wall, a balance between the activities of enzymes that generate superoxide,31 including those in the mitochondria, xanthine oxidase, cyclooxygenases, nitric oxide synthases, or NADPH oxidase (p22phox,15 p47phox [this report and Reference 1313 ], p67phox,10 and gp91phox11 ), and enzymes that scavenge superoxide or other ROS,32 including SOD, catalase, and glutathione peroxidase, determines the extent of oxidative stress in tissues. By reacting with nitric oxide, superoxide abolishes the cardioprotective functions of this molecule, which can promote development of hypertension and atherosclerosis.31 Thus, excess superoxide production, reductions in superoxide scavenger activity, or their combination can induce deleterious consequences in blood vessels.
The absolute level of superoxide liberated in the blood vessel wall may be determined by stimulation of the cellular components of this tissue. Ang II stimulated superoxide release from aortic adventitial fibroblasts in a dose-dependent manner,10 and PDGF-AB stimulated dose-dependent superoxide production in human VSMCs.20 In both cases, agonist-stimulated superoxide production was greater than that produced under basal conditions. p47phox deficiency has no apparent effect on blood pressure or atherogenesis in the progeny of apoE-/-xp47phox-/- mice; however, the roles of Ang II and PDGF were not directly addressed.33 The present results demonstrate that Ang II– or PDGF-stimulated, p47phox-dependent superoxide production by VSMCs was significantly greater than that produced under nonstimulated conditions. VSMC stimulation by these agonists may be necessary to induce ROS-mediated perturbations of cardiovascular function. In agreement with this notion, Ang II infusion concomitantly produced SOD-sensitive increases in blood pressure and aortic mRNA expression of p22phox in rats.25 Laursen et al26 showed that Ang II infusions, but not vehicle or norepinephrine infusions, in rats stimulated significant elevations in superoxide, which were accompanied by significant SOD-inhibitable increases in blood pressure. Furthermore, exposure of the canine basilar artery to graded increases in superoxide incrementally constricted this vessel in vitro.34
Together, the studies described above suggest that agonist-stimulated superoxide production may promote oxidative stress in vascular tissues, which appears to be involved in some types of hypertension2 and in atherosclerosis.35 As such, our findings demonstrate that Ang II or PDGF can stimulate a p47phox-dependent VSMC oxidase to generate significantly more superoxide than that produced under basal conditions. Future work should explore whether p47phox deficiency offers protection against hypertensive responses to chronic Ang II administration and whether the expression or activity of p47phox in VSMCs is responsive to inflammatory cytokines.
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Acknowledgments |
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The authors thank Gilda Linton for preparing the EGFP and p47phox retroviral supernatants.
Received October 25, 2000; revision received February 14, 2001; accepted March 2, 2001.
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 A. T. Baumer, H. ten Freyhaus, H. Sauer, M. Wartenberg, K. Kappert, P. Schnabel, C. Konkol, J. Hescheler, M. Vantler, and S. Rosenkranz Phosphatidylinositol 3-Kinase-dependent Membrane Recruitment of Rac-1 and p47phox Is Critical for {alpha}-Platelet-derived Growth Factor Receptor-induced Production of Reactive Oxygen Species J. Biol. Chem., March 21, 2008; 283(12): 7864 - 7876. [Abstract] [Full Text] [PDF]
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H. Choi, T. L. Leto, L. Hunyady, K. J. Catt, Y. S. Bae, and S. G. Rhee Mechanism of Angiotensin II-induced Superoxide Production in Cells Reconstituted with Angiotensin Type 1 Receptor and the Components of NADPH Oxidase J. Biol. Chem., January 4, 2008; 283(1): 255 - 267. [Abstract] [Full Text] [PDF]
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 C. I. Caldiz, C. D. Garciarena, R. A. Dulce, L. P. Novaretto, A. M. Yeves, I. L. Ennis, H. E. Cingolani, G. Chiappe de Cingolani, and N. G. Perez Mitochondrial reactive oxygen species activate the slow force response to stretch in feline myocardium J. Physiol., November 1, 2007; 584(3): 895 - 905. [Abstract] [Full Text] [PDF]
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 B. Wagner, J. M. Ricono, Y. Gorin, K. Block, M. Arar, D. Riley, G. G. Choudhury, and H. E. Abboud Mitogenic Signaling via Platelet-Derived Growth Factor beta in Metanephric Mesenchymal Cells J. Am. Soc. Nephrol., November 1, 2007; 18(11): 2903 - 2911. [Abstract] [Full Text] [PDF]
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 A. N. Lyle and K. K. Griendling Modulation of vascular smooth muscle signaling by reactive oxygen species. Physiology, August 1, 2006; 21: 269 - 280. [Abstract] [Full Text] [PDF]
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 K. Grote, M. Ortmann, G. Salguero, C. Doerries, U. Landmesser, M. Luchtefeld, R. P. Brandes, W. Gwinner, T. Tschernig, E.-G. Brabant, et al. Critical role for p47phox in renin-angiotensin system activation and blood pressure regulation Cardiovasc Res, August 1, 2006; 71(3): 596 - 605. [Abstract] [Full Text] [PDF]
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R. E. Clempus and K. K. Griendling Reactive oxygen species signaling in vascular smooth muscle cells Cardiovasc Res, July 15, 2006; 71(2): 216 - 225. [Abstract] [Full Text] [PDF]
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 L. Hunyady and K. J. Catt Pleiotropic AT1 Receptor Signaling Pathways Mediating Physiological and Pathogenic Actions of Angiotensin II Mol. Endocrinol., May 1, 2006; 20(5): 953 - 970. [Abstract] [Full Text] [PDF]
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M. Weaver, J. Liu, D. Pimentel, D. J. Reddy, P. Harding, E. L. Peterson, and P. J. Pagano Adventitial delivery of dominant-negative p67phox attenuates neointimal hyperplasia of the rat carotid artery Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1933 - H1941. [Abstract] [Full Text] [PDF]
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 H. E. Cingolani, M. C. Villa-Abrille, M. Cornelli, A. Nolly, I. L. Ennis, C. Garciarena, A. M. Suburo, V. Torbidoni, M. V. Correa, M. C. Camilionde Hurtado, et al. The Positive Inotropic Effect of Angiotensin II: Role of Endothelin-1 and Reactive Oxygen Species Hypertension, April 1, 2006; 47(4): 727 - 734. [Abstract] [Full Text] [PDF]
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 N. Ardanaz and P. J. Pagano Hydrogen peroxide as a paracrine vascular mediator: regulation and signaling leading to dysfunction. Experimental Biology and Medicine, March 1, 2006; 231(3): 237 - 251. [Abstract] [Full Text] [PDF]
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 F. Gonzalez, N. S. Rote, J. Minium, and J. P. Kirwan Reactive Oxygen Species-Induced Oxidative Stress in the Development of Insulin Resistance and Hyperandrogenism in Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., January 1, 2006; 91(1): 336 - 340. [Abstract] [Full Text] [PDF]
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 H. Crandall, Y. Ma, D. M. Dunn, R. S. Sundsbak, J. F. Zachary, P. Olofsson, R. Holmdahl, J. H. Weis, R. B. Weiss, C. Teuscher, et al. Bb2Bb3 Regulation of Murine Lyme Arthritis Is Distinct from Ncf1 and Independent of the Phagocyte Nicotinamide Adenine Dinucleotide Phosphate Oxidase Am. J. Pathol., September 1, 2005; 167(3): 775 - 785. [Abstract] [Full Text] [PDF]
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 N. R. Madamanchi, S.-K. Moon, Z. S. Hakim, S. Clark, A. Mehrizi, C. Patterson, and M. S. Runge Differential Activation of Mitogenic Signaling Pathways in Aortic Smooth Muscle Cells Deficient in Superoxide Dismutase Isoforms Arterioscler. Thromb. Vasc. Biol., May 1, 2005; 25(5): 950 - 956. [Abstract] [Full Text] [PDF]
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M. Tsuda, M. Iwai, J.-M. Li, H.-S. Li, L.-J. Min, A. Ide, M. Okumura, J. Suzuki, M. Mogi, H. Suzuki, et al. Inhibitory Effects of AT1 Receptor Blocker, Olmesartan, and Estrogen on Atherosclerosis Via Anti-Oxidative Stress Hypertension, April 1, 2005; 45(4): 545 - 551. [Abstract] [Full Text] [PDF]
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R.M. Touyz, G. Yao, M.T. Quinn, P.J. Pagano, and E.L. Schiffrin p47phox Associates With the Cytoskeleton Through Cortactin in Human Vascular Smooth Muscle Cells: Role in NAD(P)H Oxidase Regulation by Angiotensin II Arterioscler. Thromb. Vasc. Biol., March 1, 2005; 25(3): 512 - 518. [Abstract] [Full Text] [PDF]
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S. Kimura, G.-X. Zhang, A. Nishiyama, T. Shokoji, L. Yao, Y.-Y. Fan, M. Rahman, and Y. Abe Mitochondria-Derived Reactive Oxygen Species and Vascular MAP Kinases: Comparison of Angiotensin II and Diazoxide Hypertension, March 1, 2005; 45(3): 438 - 444. [Abstract] [Full Text] [PDF]
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S. H.M. Ellmark, G. J. Dusting, M. Ng Tang Fui, N. Guzzo-Pernell, and G. R. Drummond The contribution of Nox4 to NADPH oxidase activity in mouse vascular smooth muscle Cardiovasc Res, February 1, 2005; 65(2): 495 - 504. [Abstract] [Full Text] [PDF]
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R. P. Brandes and J. Kreuzer Vascular NADPH oxidases: molecular mechanisms of activation Cardiovasc Res, January 1, 2005; 65(1): 16 - 27. [Abstract] [Full Text] [PDF]
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A. Segev, N. Nili, and B. H Strauss The role of perlecan in arterial injury and angiogenesis Cardiovasc Res, September 1, 2004; 63(4): 603 - 610. [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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 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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 R. S. Frey and A. B. Malik Oxidant signaling in lung cells Am J Physiol Lung Cell Mol Physiol, January 1, 2004; 286(1): L1 - L3. [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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H. Hua, S. Munk, H. Goldberg, I. G. Fantus, and C. I. Whiteside High Glucose-suppressed Endothelin-1 Ca2+ Signaling via NADPH Oxidase and Diacylglycerol-sensitive Protein Kinase C Isozymes in Mesangial Cells J. Biol. Chem., September 5, 2003; 278(36): 33951 - 33962. [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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J.-M. Li and A. M. Shah ROS Generation by Nonphagocytic NADPH Oxidase: Potential Relevance in Diabetic Nephropathy J. Am. Soc. Nephrol., August 1, 2003; 14(90003): S221 - 226. [Abstract] [Full Text] [PDF]
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 M. Geiszt, K. Lekstrom, S. Brenner, S. M. Hewitt, R. Dana, H. L. Malech, and T. L. Leto NAD(P)H Oxidase 1, a Product of Differentiated Colon Epithelial Cells, Can Partially Replace Glycoprotein 91phox in the Regulated Production of Superoxide by Phagocytes J. Immunol., July 1, 2003; 171(1): 299 - 306. [Abstract] [Full Text] [PDF]
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R. Takeya, N. Ueno, K. Kami, M. Taura, M. Kohjima, T. Izaki, H. Nunoi, and H. Sumimoto Novel Human Homologues of p47phox and p67phox Participate in Activation of Superoxide-producing NADPH Oxidases J. Biol. Chem., June 27, 2003; 278(27): 25234 - 25246. [Abstract] [Full Text] [PDF]
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 K. Grote, I. Flach, M. Luchtefeld, E. Akin, S. M. Holland, H. Drexler, and B. Schieffer Mechanical Stretch Enhances mRNA Expression and Proenzyme Release of Matrix Metalloproteinase-2 (MMP-2) via NAD(P)H Oxidase-Derived Reactive Oxygen Species Circ. Res., June 13, 2003; 92 (11): e80 - e86. [Abstract] [Full Text] [PDF]
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 D. G Harrison, Hua Cai, U. Landmesser, and K. K Griendling The Pickering Lecture British Hypertension Society, 10th September 2002: Interactions of angiotensin II with NAD(P)H oxidase, oxidant stress and cardiovascular disease Journal of Renin-Angiotensin-Aldosterone System, June 1, 2003; 4(2): 51 - 61. [Abstract] [PDF]
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M. E. Cifuentes and P. J. Pagano c-Src and Smooth Muscle NAD(P)H Oxidase: Assembling a Path to Hypertrophy Arterioscler. Thromb. Vasc. Biol., June 1, 2003; 23(6): 919 - 921. [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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B. Banfi, R. A. Clark, K. Steger, and K.-H. Krause Two Novel Proteins Activate Superoxide Generation by the NADPH Oxidase NOX1 J. Biol. Chem., January 31, 2003; 278(6): 3510 - 3513. [Abstract] [Full Text] [PDF]
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F. E. Rey and P. J. Pagano The Reactive Adventitia: Fibroblast Oxidase in Vascular Function Arterioscler. Thromb. Vasc. Biol., December 1, 2002; 22(12): 1962 - 1971. [Abstract] [Full Text] [PDF]
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A. Tojo, M. L. Onozato, N. Kobayashi, A. Goto, H. Matsuoka, and T. Fujita Angiotensin II and Oxidative Stress in Dahl Salt-Sensitive Rat With Heart Failure Hypertension, December 1, 2002; 40(6): 834 - 839. [Abstract] [Full Text] [PDF]
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 K. SATO, M. B. KADIISKA, A. J. GHIO, J. CORBETT, Y. C. FANN, S. M. HOLLAND, R. G. THURMAN, and R. P. MASON In vivo lipid-derived free radical formation by NADPH oxidase in acute lung injury induced by lipopolysaccharide: a model for ARDS FASEB J, November 1, 2002; 16(13): 1713 - 1720. [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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P. N. Seshiah, D. S. Weber, P. Rocic, L. Valppu, Y. Taniyama, and K. K. Griendling Angiotensin II Stimulation of NAD(P)H Oxidase Activity: Upstream Mediators Circ. Res., September 6, 2002; 91(5): 406 - 413. [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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