Published online before print January 3, 2006, doi: 10.1161/01.HYP.0000200023.02195.73
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(Hypertension. 2006;47:238.)
© 2006 American Heart Association, Inc.
Original Articles |
RNA Silencing In Vivo Reveals Role of p22phox in Rat Angiotensin Slow Pressor Response
From the Division of Cardiology (D.G.H., K.K.G.), Emory University, Atlanta, Ga; Angiogenesis Section (P.S.G., A.W., Y.C., W.J.W., C.W.), Lombardi Cancer Center, and Cardiovascular-Kidney Institute and Division of Nephrology and Hypertension (P.M., T.C., P.S.G., M.M., M.L., J.R., Y.C., W.J.W., C.S.W.) Georgetown University, Washington, DC.
Correspondence to Christopher S. Wilcox, Georgetown University Medical Center–PHC F6003, 3800 Reservoir Rd, NW, Washington, DC 20007. E-mail wilcoxch{at}georgetown.edu
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Abstract |
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The angiotensin II (Ang II) slow-pressor response entails an increase in mean arterial pressure and reactive oxygen species. We used double-stranded interfering RNAs (siRNAs) in Sprague Dawley rats in vivo to test the hypothesis that an increase in the p22phox component of NADPH oxidase is required for this response. Reactive oxygen species were assessed from excretion of 8-isoprostane prostaglandin F2
and blood pressure by telemetry. Two siRNA sequences to p22phox (sip22phox) reduced mRNA >85% in cultured vascular smooth muscle cells. Rats received rapid (10 second) IV injections (50 to 100 µg) of 1 of 2 different sip22phox, control siRNA, or vehicle (TransIt in saline) during 14 day SC infusions of Ang II (200 ng · kg–1 · min–1) or sham infusions. In both groups, sip22phox, relative to control siRNA, led to significant (P<0.001; 
50%) reductions in expression of p22phox mRNA and protein and of NADPH oxidase activity in the kidney cortex. In Ang II–infused rats, sip22phox decreased protein expression for Nox-1, -2, and -4 but increased p47phox. Three days after sip22phox, conscious rats infused with Ang II had a reduced excretion of 8-isoprostane (10±1 versus 19±2 pg · 24 h–1; P<0.01) and a reduced mean arterial pressure (142±5 versus 168±4 mm Hg; P<0.005). An increase in p22phox is required for increased renal NADPH oxidase activity, expression of Nox proteins and oxidative stress, and contributes 
50% to hypertension during an Ang II slow-pressor response.
Key Words: hypertension, arterial • arterioles • oxidative stress • kidney
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Introduction |
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Angiotensin II (Ang II) has been assigned a critical role in the generation and complications of human essential hypertension, yet plasma renin activity and plasma concentrations of Ang II are not remarkably elevated.1 Mice,2 rats,3,4 rabbits,5 or humans6 infused with Ang II at doses that are initially subpressor develop a "slow-pressor response" in which the blood pressure (BP) increases progressively despite plasma Ang II concentrations that are increased only moderately.3 The kidney is implicated in the slow-pressor response, because the development of hypertension depends on salt intake.3 Moreover, rats or rabbits infused with Ang II have enhanced renal vasoconstriction to Ang II5,7 despite downregulation of Ang II type 1 receptors.
Reactive oxygen species (ROS) and superoxide anion (O2·–) have been implicated in the development of hypertension in the Ang II slow-pressor model, because hypertension is prevented by antioxidant molecules, such as a permeabilized form of superoxide dismutase or tempol, which is an superoxide dismutase mimetic nitroxide.2,4,5,8 Infusions of Ang II increase the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in blood vessels9 and the kidney cortex.4,10,11 This complex enzyme, which was first described in phagocytes and, later, in blood vessels and the kidney, is composed of membrane-associated components of the flavoprotein catalytic core, gp91phox (now named Nox-2) and p22phox.12 Activation requires phosphorylation of p47phox13 and its assembly with p67phox14 and Rac-1 at the membrane.12 Homologues of Nox-2 include Nox-1, which has been characterized in vascular smooth muscle cells (VSMCs),9 and Nox-4, which has been characterized in the kidney.15 VSMCs and kidneys have the phagocytic NADPH oxidase components.12 p22phox is expressed in the thick ascending limb, macula densa, distal convoluted tubule, collecting ducts, vasculature, and interstitial fibroblasts of the kidney.16 It is believed to dock the enzyme complex in the cell membrane and stabilize Nox proteins.12 There is colocalization of p22phox and O2·– generation in atherosclerotic plaques from human blood vessels.17 The p22phox component is upregulated in the kidneys4,10 of rats undergoing an Ang II slow-pressor response. Antisense constructs targeted at p22phox inhibit Ang II–induced hypertrophy of cultured VSMCs,18 and mice overexpressing p22phox in their VSMCs have an exaggerated hypertrophic response to Ang II infusion.19
Whereas these observations demonstrate the importance of p22phox in mediating the effects of Ang II on ROS and development of atherosclerosis, recent studies with mice overexpressing p22phox in blood vessels show that, despite increased aortic expression of p22phox and Nox-1 and increased O2·– and H2O2 generation, the basal BP is not increased20 and rises only slightly more rapidly during infusion of Ang II.19 Therefore, the role of p22phox in the physiological response to Ang II is not yet clear. We tested the hypothesis that an increase in p22phox is required for increased NADPH oxidase activity and development of oxidative stress and hypertension during an Ang II slow-pressor response. Because specific pharmacological inhibitors or knockouts for p22phox are not yet available, we used an RNA interference (RNAi) strategy with small (21-bp) double-stranded interfering RNAs (siRNAs)21,22 targeted to p22phox (sip22phox) to test its role in mediating responses to Ang II infusion in rats.
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Methods |
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Animal Preparation and Studies of Isoprostane Excretion, mRNA, and Protein Expression
Studies were approved by the Georgetown University Animal Care and Use Committee. Experiments were performed on male Sprague-Dawley rats weighing 280 to 350 g maintained on a synthetic casein–based diet with precisely regulated NaCl content (Na+ 0.3 g · 100 g–1; Teckland Inc, Madison, WI) as described.23 Rats were maintained in individual cages under conditions of constant temperature and humidity and exposed to 12-hour cycles of light and dark with unrestricted water intake.
Under brief (10 minutes) anesthesia with 1% to 2% isoflurane, an osmotic minipump (model 2002, Alzet Corporation) was inserted subcutaneously in the nape of the neck and filled with vehicle (sham infused) or human Ang II (Peninsula Laboratories, San Carlos, CA) to deliver 200 ng–1 · kg–1 · min–1 (Ang II–infused). This protocol provides a gradual increase in mean arterial pressure (MAP) with Ang II over 2 weeks4 with increases in excretion of 8-isoprostane (8-Iso) prostaglandin F (PGF)2 and malondialdehyde and increased expression of p22phox mRNA10 and protein4 in the kidney. Under the same anesthetic, a cannula was inserted into a femoral vein, tunneled subcutaneously to the nape of the neck, filled with heparin saline, and plugged. This was used subsequently for intravenous injections of siRNA or vehicle.
The aim of the first series was to assess the effects of an infusion of Ang II on renal expression of mRNA and protein for p22phox, renal NADPH oxidase activity, excretion of 8-isoprostane PGF2, and MAP. Eleven days after insertion of minipumps containing vehicle or Ang II, rats were placed in metabolic cages, and on day 12, a 24-hour urine was collected for excretion of 8-Iso. The next day, rats were euthanized for harvesting kidneys. Separate groups of rats were equipped with telemetric BP recorders (see below) and, after 2 weeks for recovery, received sham or Ang II infusions. MAP was recorded on day 12.
The aim of the second series was to assess the effects of sip22phox on the renal cortical expression of mRNA and protein for p22phox and NADPH oxidase activity. Rats received 2 injections, one of 100 µg of siRNA (or equivalent vehicle) on day 5 of Ang II or sham infusions and another of 50 µg on day 8. The siRNA was complexed with a polymer from TransIT in vivo gene delivery system and delivered according to the manufacturer’s recommendations (Mirus Inc). The injections of 6 mL were given via a catheter in a jugular vein over 10 seconds. Rats were euthanized 72 hours after the second injection of siRNA. Under thiobarbital anesthesia (Inactin; 100 mg · kg–1 IP; Research Biochemicals Inc), the kidneys were flushed free of blood with PBS, harvested, and the cortex dissected and frozen for subsequent analysis of mRNA, protein, and NADPH oxidase activity.
The aim of the third series was to assess the effects of sip22phox on either the excretion of 8-Iso or the MAP during the hypertensive phase of an Ang II slow-pressor response. One group was infused with Ang II (n=6 to 8 per group) and was habituated to metabolic cages on day 7 of Ang II infusion. Twenty-four–hour urine collections were undertaken on day 8 (before intravenous injections) and daily until day 13 of Ang II infusion for excretion of 8-Iso, as described previously.10 On day 9, rats received 6 mL of fluid IV containing either a vehicle or 50 µg of a control siRNA (siCont) or sip22phox given by rapid IV bolus over 10 s as in series 2.24 The complex of siRNA and TransIt were incubated together for 5 minutes before injection. A second group was briefly anesthized for insertion of BP telemeters (see below) and, after 14 days for recovery, was infused with Ang II and given 6 mL IV injections of siRNA or vehicle on days 5 and 8 as in the second series. This protocol was adopted from studies that had used in vivo siRNA successfully to target genes in mice21,24 and on results of pilot studies (see beginning of Results section).
The aim of the fourth group was to investigate potential off-target effects of siRNA in Ang II–infused rats.25 Rats were given intravenous injections of sip22phox No. 2 targeted to a different part of the molecule (see below). Its effects on mRNA and protein expression for p22phox and NADPH oxidase activity were compared with sip22phox No. 1. The kidney cortex was also analyzed for mRNA expression of toll-like receptor 3 (TLR-3) and signal transducers and activators transcription-1 (STAT-1) to assess activation of the interferon (INF) response element pathway that can confound siRNA studies.26
An extended Methods section is available online at http://hyper. ahajournals.org for the following subsections: telemetric BP recording and injection of siRNAs; siRNA construction and validation; mRNA isolation and RT-PCR; protein isolation, quantification, and immunoblotting; NADPH oxidase activity; chemical methods; and statistical analysis.
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Results |
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Pilot studies of isoprostane excretion of rats infused with Ang II were undertaken to establish optimal intravenous doses of sip22phox constructs. Two days after 5-µg sip22phox, the excretion of 8-Iso was reduced by 20±10% (n=6), whereas 50 µg reduced excretion more consistently and significantly by 50% (see below). A dose of 200 µg produced more variable results. siCont had no effect. Maximal effects were apparent at 36 to 72 hours after injection. Therefore, a dose of 50 to 100 µg was used, and organs were harvested 72 hours after injection.
In series I, we found that 12 days of Ang II infusion increased the MAP, NADPH oxidase activity, 8-Iso excretion, and p22phox mRNA and protein in the renal cortex (Figure 1). In series 2, we found that sip22phox injection reduced the renal p22phox mRNA and protein by 50% and had a similar effect on NADPH oxidase activity both in sham-infused (Figure 2 A) in Ang II–infused rats (Figure 2B).
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; n=8) with Ang II infusions (
; n=8) on renal cortical expression for p22phox mRNA (A) or protein (B), NADPH-stimulated superoxide anion generation (C), 24-hour excretion of 8-iso PGF2
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), or sip22phox ( 
) in sham-infused (A) or Ang II–infused (B) rats. Compared with vehicle: *P<0.05; **P<0.01; ***P<0.005.
Before injection of vehicle or siRNA, the 8-isoprostane PGF2 excretion of rats infused with Ang II averaged 12.5±1.8 pg · 24 h–1. It increased progressively over 4 days in Ang II–infused rats that received a vehicle or siCont but not in those receiving sip22phox (Figure 3 A). This resulted in a 50% (P<0.01) reduction in excretion of 8-isoprostane PGF2 in the group receiving sip22phox, relative to siCont or vehicle, from 48 to 96 hours after injection.
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and continuous lines), siCont (• and continuous lines), or sip22phox (
and broken lines). Compared with siCont: *P<0.05; **P<0.01; ***P<0.005.
Ang II caused progressive increases in MAP after the first day of infusion (Figure 3B) to a plateau by day 9 of 170 mm Hg in rats receiving injections of vehicle or siCont. In contrast, there was no significant increase in MAP in those receiving sip22phox from the time of the first injection (day 5; 132±3 mm Hg) to 72 hours after the second injection (day 11; 140±5 mm Hg). There were no differences in heart rate between the groups 3 days after injections (vehicle: 339±14; siCont: 378±28; sip22phox No. 1: 355±11; sip22phox No. 2: 358±9 min–1; P value not significant). The MAP of Ang II–induced rats given sip22phox remained significantly (P<0.005) above levels of sham-infused rats of 107±4 mm Hg. Therefore, an increase in p22phox expression and activity in the kidney can account for up to about one third of the increase in BP during a 12-day infusion of Ang II.
The renal cortical expression of the other NADPH oxidase proteins in angiotensin-infused rats is depicted in Figure 4. There were no differences in expression between rats receiving vehicle and siCont, but sip22phox significantly reduced the expression of Nox-1, -2, and -4 and increased the expression of p47phox.
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As shown in Figure 5, the sip22phox No. 1 and No. 2 (targeted to different regions of p22phox cDNA) produced equivalent reductions in p22phox mRNA and protein expression and MAP. There were also equivalent reductions in NADPH oxidase activity (siCont: 54±7; sip22phox No. 1: 29±4; sip22phox No. 2: 30±5, 103 counts · mg protein–1 · 5 min–1). Therefore, data for sip22phox No. 1 and No. 2 are presented together.
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), or sip22phox #2 (siRNA administration can activate off-target effects via cellular signaling through the TLR-3 with synthesis of interferons that signal via STAT-1. We found no significant increases in the expression of these mRNAs in the kidney cortex after injections of siCont or sip22phox, relative to vehicle, in Ang II–infused rats (Figure 6), indicating that such off-target effects of our sip22phox were minimal or absent.
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Discussion |
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We confirm that Ang II infusion causes a progressive increase in MAP,2,3 accompanied by increased expression of mRNA and protein for p22phox in the kidneys,5,10 increased renal cortical NADPH oxidase activity,11 and increased excretion of the lipid peroxidation marker 8-isoprostane PGF2
.5,10 This study demonstrates the feasibility of "gene knockdown" in vivo in rats with a simple RNAi strategy using an RNA complexing solution and a rapid, relatively large-volume injection. This prevented increased expression of a target mRNA and protein in the kidney and thereby provided a model to investigate its biochemical and physiological actions. The main new findings are that injection of sip22phox prevents an increase in expression of p22phox mRNA and protein in the kidney and prevents an increase in NADPH oxidase activity, oxidative stress, and progressive rise in MAP of conscious rats during the second week of an Ang II infusion. In contrast, injection of a control siRNA sequence did not perturb these parameters. The effect is apparent between 48 and 96 hours after injection and was seen with 2 siRNAs targeted to different regions of the p22phox cDNA. The reduction in renal cortical expression of p22phox was accompanied by reduced expression of Nox-1, -2, and -4 but increased expression of p47phox.
RNAi originated with the finding that double-stranded RNAs injected into Caenorhabditis elegans silenced genes with complementary sequences.22 Double-stranded RNA can be processed in cells into siRNAs, 20 to 22 nucleotides in length, by the enzyme Dicer,27 and are incorporated into an RNA-induced silencing complex, which silences complimentary RNAs.21,27 Hydrodynamic transfection of siRNAs in mice involves rapid injection of a sufficiently large volume to transiently increase venous pressure24 to silence genes in organs with high blood flow, including the kidney28 and blood vessel.28 A modified strategy of rapid IV injection over 10 s of siRNAs in a gene complexing solution of TransIt in saline was effective in reducing the expression of the target mRNA and protein in vivo in the kidney cortex. The sip22phox injection led to a 50% reduction in p22phox expression in the kidneys, matched by an equivalent reduction in the target enzyme activity in both sham and Ang II–infused rats. Apparently, the increase in NADPH oxidase activity in the kidney with Ang II depends on increased p22phox protein expression. Additional studies will be required to determine whether similar effects of RNAi can be achieved in rats with this method in other organs besides the kidney and whether improved delivery or more stable constructs can increase the efficiency and prolong the duration of the therapeutic effects.
Potential problems with in vivo RNAi have been reviewed.27 First, at high concentration, some siRNAs may act via TLR-3 to increase the INF response factor that stimulates INF and initiates cell signaling via STAT-1.26 Therefore, we undertook pilot studies in Ang II–infused rats to define a minimally effective dose of siRNA. We confirmed that siRNA injections did not change the renal mRNA expression for TLR-3 or STAT-1 in angiotensin-infused rats. Thus, it seems unlikely that activation of this pathway could account for our results. Second, there are off-target effects of RNAi because of silencing of genes with as few as 11 contiguous nucleotides of identity to the siRNA.25 Therefore, we limited the quantity of siRNA injected to 200 to 400 µg · kg–1 (below the quantities used previously in mice28), tested a control, nonsilencing sequence, and found that 2 active siRNAs targeted to different coding regions of the p22phox gene were equally effective in reducing p22phox expression, NADPH oxidase activity, and BP in Ang II–infused rats. Thus, the responses observed likely relate to reductions in p22phox mRNA and protein expression.
ROS have been assigned a critical role in vasoconstriction,11 endothelial dysfunction,5 vascular remodeling,19 and atherosclerosis17 accompanying hypertension. In the kidney, ROS can enhance many processes linked to the development of hypertension, including tubuloglomerular feedback,29 afferent arteriolar vasoconstriction,11 NaCl reabsorption,30 and oxygen usage.23 Ang II can upregulate many of the components of NADPH oxidase in addition to p22phox in blood vessels or kidneys, including Nox-19,10, Nox-2,31 p67phox,31 and p47phox.13 Mice with targeted deletions of p47phox have a diminished increase in BP with Ang II.13 A Nox-2–blocking peptide reduces Ang II–induced hypertension in rats,32 whereas a Nox-2 knockout mouse has a reduced basal BP but a normal increase in BP with a full dose of Ang II despite a failure to increase aortic production of O2·–.33 The relative importance of Nox-1, -2, and -4 in mediating effects of Ang II requires additional study. p22phox is expressed widely in the normal rat kidney in cells of the thick ascending limb of the loop of Henle, macula densa, distal tubule, cortical outer medullary and inner medullary collecting ducts, and vasculative and interstitial fibroblasts.16 The present studies demonstrate that upregulation of p22phox can account for about one third of the increase in BP, all of the increase in NADPH oxidase activity in the renal cortex, and the progressive increase in isoprostane excretion during an Ang II infusion.
Limitations of the present study include a lack of mechanism to explain how a reduction in p22phox expression leads to a reduction in BP during Ang II infusion. Additional studies are needed to document the cellular sites of changes in p22phox in resistance arterioles in systemic vessels and kidneys, in renal tubular epithelial cells and, perhaps, inflammatory cells, and the effects of these on salt transport, renin secretion, and vascular reactivity. A second limitation is that studies of NADPH oxidase expression and activity were confined to the kidneys. It is not clear to what extent p22phox was reduced at other sites and the relative contributions of renal and extrarenal changes in p22phox to the results obtained.
Perspectives
The p22phox component interacts directly with Nox-1, -2, and -4 proteins in VSMCs to form a functional O2·– generating NADPH oxidase.33,34 The reductions in Nox-1, -2, and -4 expression in rats with the p22phox gene silenced may explain the reduced NADPH oxidase activity. Nox-2 is the prominent Nox isoform expressed in human resistance vessels.35 Nox-1 is prominent in conduit vessels,9 and Nox-4 is expressed strongly in the kidneys.15 Because the resistance vessels and kidneys are prominent sites for Ang II–induced hypertension, this may explain the moderation of hypertension by silencing of p22phox during Ang II infusion. Presently, there is no way to selectively prevent activation of NADPH oxidase. Drugs, such as diphenyliodinium, inhibit a wide range of flavin-containing enzymes in addition to NADPH oxidase. Pyocynin inhibits the activation of Nox-2 by p47phox but p47phox is not required for activation of Nox-1 or Nox-4.36 Thus, p22phox appears to be the only unique requirement for activity of all Nox enzymes.34 This strategy of p22phox silencing may have use in experimental studies and clinical settings in which a specific knockdown of all Nox enzymes is desirable. This study has, indeed, demonstrated that p22phox plays a critical role in enhancing the expression of catalytic Nox components of NADPH oxidase during Ang II and may, therefore, be a useful target for drug development to obviate oxidative stress and its consequences in the hypertensive kidney.
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Acknowledgments |
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This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-36079 and DK-49870), the National Heart, Lung, and Blood Institute (HL-68686), and by funds from the George E. Schreiner Chair of Nephrology. We thank Margaret Brierton for preparing the article.
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Footnotes |
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P.M., T.C., and P.S.G. contributed equally to this study.
Dr Wilcox has a research grant from Novartis, GSK, and research support from Cary Pharmaceuticals. He is also a consultant/advisory board member for Berlex, Nitromed, Davita, Pfizer, and Cary Pharmaceuticals.
Received September 28, 2005; first decision October 21, 2005; accepted December 5, 2005.
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References |
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1. Brown JJ, Davies DL, Lever AF, Robertson JIS. Plasma renin concentration in human hypertension. 1: Relationship between renin, sodium, and potassium. BMJ. 1965; 2: 144–148.
2. Kawada N, Imai E, Karber A, Welch WJ, Wilcox CS. A mouse model of angiotensin II slow pressor response: role of oxidative stress. J Am Soc Nephrol. 2002; 13: 2860–2868.
3. Brown AJ, Casals-Stenzel J, Gofford S, Lever AF, Morton JJ. Comparison of fast and slow pressor effects of angiotensin II in the conscious rats. Am J Physiol. 1981; 241: H381–H388.[Medline] [Order article via Infotrieve]
4. Welch WJ, Blau J, Xie H, Chabrashvili T, Wilcox CS. Angiotensin-induced defects in renal oxygenation: role of oxidative stress. Am J Physiol. 2004; 288: H22–H28.
5. Wang D, Chabrashvili T, Wilcox CS. Enhanced contractility of renal afferent arterioles from angiotensin-infused rabbits: roles of oxidative stress, thromboxane-prostanoid receptors and endothelium. Circ Res. 2004; 94: 1436–1442.
6. Ames RP, Borkowski AJ, Sicinski AM, Laragh JH. Prolonged infusions of angiotensin II and norepinephrine and blood pressure, electrolyte balance, aldosterone and cortisol secretion in normal man and in cirrhosis with ascites. J Clin Invest. 1965; 44: 1171–1186.[Medline] [Order article via Infotrieve]
7. Imig JD. Afferent arteriolar reactivity to angtiotensin II is enhanced during the early phase of angiotensin II hypertension. Am J Hypertens. 2000; 13: 810–818.[CrossRef][Medline] [Order article via Infotrieve]
8. 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.
9. Lassegue B, Sorescu D, Szocs 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.
10. Chabrashvili T, Kitiyakara C, Blau J, Karber A, Aslam S, Welch WJ, Wilcox CS. Effects of Ang II type 1 and 2 receptors on oxidative stress, renal NADPH oxidase and SODs expression. Am J Physiol. 2003; 285: R117–R124.
11. Wang D, Chen Y, Chabrashvili T, Aslam S, Borrego L, Umans J, Wilcox CS. Role of oxidative stress in endothelial dysfunction and enhanced responses to Ang II of afferent arterioles from rabbits infused with Ang II. J Am Soc Nephrol. 2003; 14: 2783–2789.
12. Lassegue B, Clempus RE. Vascular NAD(P)H oxidases: specific features, expression, and regulation. Am J Physiol. 2003; 285: R277–R297.
13. Landmesser U, Cai H, Dikalov S, McCann L, Hwang J, Jo H, Holland SM, Harrison DG. Role of p47(phox) in vascular oxidative stress and hypertension caused by angiotensin II. Hypertension. 2002; 40: 511–515.
14. Pagano PJ, Chanock SJ, Siwik DA, Colucci WS, Clark JK. Angiotensin II induces p67phox mRNA expression and NADPH oxidase superoxide generation in rabbit aortic adventitial fibroblasts. Hypertension. 1998; 32: 331–337.
15. Geiszt M, Kopp JB, Várnai P, Leto TL. Identification of renox, an NADPH oxidase in kidney. Proc Natl Acad Sci U S A. 2000; 97: 8010–8014.
16. Chabrashvili T, Tojo A, Onozato M, Kitiyakara C, Quinn MT, Fujita T, Welch WJ, Wilcox CS. Expression and cellular localization of classic NADPH oxidase subunits in the spontaneously hypertensive rat kidney. Hypertension. 2002; 39: 269–274.
17. Sorescu D, Weiss D, Lassegue B, Clempus RE, Szocs K, Soreescu GP, Valppu L, Quinn MT, Lambeth JD, Vega JD, Taylor R, Griendling KK. Superoxide production and expression of Nox family proteins in human atherosclerosis. Circulation. 2002; 105: 1429–1435.
18. Ushio-Fukai M, Zafari AM, Fukui T, Ishizaka N, Griendling KK. p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin II-induced hypertrophy in vascular smooth muscle cells. J Biol Chem. 1996; 271: 23317–23321.
19. Weber DS, Rocic P, Mellis AM, Laude KM, Lyle A, Harrison DG, Griendling KK. Angiotensin II-induced hypertrophy is potentiated in mice overexpressing p22phox in vascular smooth muscle. Am J Physiol. 2004; 288: H37–H42.
20. Laude KM, Cai H, Fink B, Hoch N, Weber DS, McCann L, Kojda G, Fukai T, Dikalov S, Ramasamy S, Gamez G, Griendling KK, Harrison DG. Hemodynamic and biochemical adaptations to vascular smooth muscle overexpression of p22phox in mice. Am J Physiol. 2004; 288: H7–H12.
21. Mello CC, Conte D. Revealing the world of RNA interference. Nature. 2004; 431: 338–342.[CrossRef][Medline] [Order article via Infotrieve]
22. Fire A, Albertson D, Harrison S, Moerman DG. Production of antisense RNA leads to effective and specific inhibition of gene expression in C. elegans muscle. Development. 1991; 113: 503–514.[Abstract]
23. Welch WJ, Blau J, Xie H, Chabrashvili T, Wilcox CS. Angiotensin-induced defects in renal oxygenation: role of oxidative stress. Am J Physiol. 2005; 288: H22–H28.
24. Liu F, Song YK, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Therapy. 1999; 6: 1258–1266.[CrossRef][Medline] [Order article via Infotrieve]
25. Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M, Li B, Carvet G, Linsley PS. Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol. 2003; 21: 635–637.[CrossRef][Medline] [Order article via Infotrieve]
26. Bridge AJ, Pebernard S, Ducraux A, Nicoulaz AL, Iggo R. Induction of an interferon response by RNAi vectors in mammalian cells. Nature Genet. 2003; 34: 263–264.[CrossRef][Medline] [Order article via Infotrieve]
27. Hannon GJ, Rossi JJ. Unlocking the potential of the human genome with RNA interference. Nature. 2004; 431: 371–378.[CrossRef][Medline] [Order article via Infotrieve]
28. Hamar P, Song E, Kökény G, Chen A, Ouyang N, Lieberman J. Small interfering RNA targeting Fas protects mice against renal ischemia-reperfusion injury. Proc Natl Acad Sci U S A. 2004; 101: 14883–14888.
29. Welch WJ, Tojo A, Wilcox CS. Roles of NO and oxygen radicals in tubuloglomerular feedback in SHR. Am J Physiol. 2000; 278: F769–F776.
30. Majid DS, Nishiyama A. Nitric oxide blockade enhances renal responses to superoxide dismutase inhibition in dogs. Hypertension. 2002; 39: 293–297.
31. 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.
32. Rey FE, Li XC, Carretero OA, Garvin JL, Pagano PJ. Perivascular superoxide anion contributes to impairment of endothelium-dependent relaxation: role of gp91(phox). Circulation. 2002; 106: 2497–2502.
33. Wang HD, Xu S, Johns DG, Du Y, Quinn MT, Cayatte AJ, Cohen RA. Role of NADPH oxidase in the vascular hypertrophic and oxidative stress response to angiotensin II in mice. Circ Res. 2001; 88: 947–953.
34. Ambasta RK, Kumar P, Griendling KK, Schmidt HHW, Busse R, Brandes RP. Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase. J Biol Chem. 2004; 279: 45935–45941.
35. Touyz RM, Chen X, Tabet F, Yao G, He G, Quinn MT, Pagano PJ, Schiffrin EL. 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. 2002; 90: 1205–1213.
36. Sumimoto H, Ueno N, Yamasaki T, Taura M, Takeya R. Molecular mechanism underlying activation of superoxide-producing NADPH oxidases: roles for their regulatory proteins. Jpn J Infect Dis. 2004; 57: S24–S25.[Medline] [Order article via Infotrieve]
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 S. J. Wilson, C. C. Cavanagh, A. M. Lesher, A. J. Frey, S. E. Russell, and E. M. Smyth Activation-dependent stabilization of the human thromboxane receptor: role of reactive oxygen species J. Lipid Res., June 1, 2009; 50(6): 1047 - 1056. [Abstract] [Full Text] [PDF]
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 R. Zhang, P. Harding, J. L. Garvin, R. Juncos, E. Peterson, L. A. Juncos, and R. Liu Isoforms and Functions of NAD(P)H Oxidase at the Macula Densa Hypertension, March 1, 2009; 53(3): 556 - 563. [Abstract] [Full Text] [PDF]
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 C. S. Wilcox and A. Pearlman Chemistry and Antihypertensive Effects of Tempol and Other Nitroxides Pharmacol. Rev., December 1, 2008; 60(4): 418 - 469. [Abstract] [Full Text] [PDF]
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G. B. Silva and J. L. Garvin Angiotensin II-Dependent Hypertension Increases Na Transport-Related Oxygen Consumption by the Thick Ascending Limb Hypertension, December 1, 2008; 52(6): 1091 - 1098. [Abstract] [Full Text] [PDF]
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 S. Kelsen, B. J. Patel, L. B. Parker, T. Vera, J. M. Rimoldi, R. S. V. Gadepalli, H. A. Drummond, and D. E. Stec Heme oxygenase attenuates angiotensin II-mediated superoxide production in cultured mouse thick ascending loop of Henle cells Am J Physiol Renal Physiol, October 1, 2008; 295(4): F1158 - F1165. [Abstract] [Full Text] [PDF]
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R. A. Gonzalez-Villalobos, D. M. Seth, R. Satou, H. Horton, N. Ohashi, K. Miyata, A. Katsurada, D. V. Tran, H. Kobori, and L. G. Navar Intrarenal angiotensin II and angiotensinogen augmentation in chronic angiotensin II-infused mice Am J Physiol Renal Physiol, September 1, 2008; 295(3): F772 - F779. [Abstract] [Full Text] [PDF]
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W. J. Welch Angiotensin II-Dependent Superoxide: Effects on Hypertension and Vascular Dysfunction Hypertension, July 1, 2008; 52(1): 51 - 56. [Full Text] [PDF]
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 T. M. Paravicini and R. M. Touyz NADPH Oxidases, Reactive Oxygen Species, and Hypertension: Clinical implications and therapeutic possibilities Diabetes Care, February 1, 2008; 31(Supplement_2): S170 - S180. [Abstract] [Full Text] [PDF]
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 C. F.M.Z. Clemente, T. F. Tornatore, T. H. Theizen, A. C. Deckmann, T. C. Pereira, I. Lopes-Cendes, J. R. M. Souza, and K. G. Franchini Targeting Focal Adhesion Kinase With Small Interfering RNA Prevents and Reverses Load-Induced Cardiac Hypertrophy in Mice Circ. Res., December 7, 2007; 101(12): 1339 - 1348. [Abstract] [Full Text] [PDF]
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D. Wang, P. S. Gill, T. Chabrashvili, M. L. Onozato, J. Raggio, M. Mendonca, K. Dennehy, M. Li, P. Modlinger, J. Leiper, et al. Isoform-Specific Regulation by NG,NG-Dimethylarginine Dimethylaminohydrolase of Rat Serum Asymmetric Dimethylarginine and Vascular Endothelium-Derived Relaxing Factor/NO Circ. Res., September 14, 2007; 101(6): 627 - 635. [Abstract] [Full Text] [PDF]
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 A. Sachse and G. Wolf Angiotensin II Induced Reactive Oxygen Species and the Kidney J. Am. Soc. Nephrol., September 1, 2007; 18(9): 2439 - 2446. [Abstract] [Full Text] [PDF]
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 P. Nouri, P. Gill, M. Li, C. S. Wilcox, and W. J. Welch p22phox in the macula densa regulates single nephron GFR during angiotensin II infusion in rats Am J Physiol Heart Circ Physiol, April 1, 2007; 292(4): H1685 - H1689. [Abstract] [Full Text] [PDF]
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W. J. Welch, T. Chabrashvili, G. Solis, Y. Chen, P. S. Gill, S. Aslam, X. Wang, H. Ji, K. Sandberg, P. Jose, et al. Role of Extracellular Superoxide Dismutase in the Mouse Angiotensin Slow Pressor Response Hypertension, November 1, 2006; 48(5): 934 - 941. [Abstract] [Full Text] [PDF]
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