Endothelin-1 Increases Vascular Superoxide via EndothelinA–NADPH Oxidase Pathway in Low-Renin Hypertension Lixin Li, MD; Gregory D. Fink, PhD; Stephanie W. Watts, PhD; Carrie A. Northcott, MS; James J. Galligan, PhD; Patrick J. Pagano, PhD; Alex F. Chen, MD, PhD Background—Angiotensin II–induced hypertension is associated with NAD(P)H oxidase– dependent superoxide production in the vessel wall. Vascular superoxide level is also increased in deoxycorticosterone acetate (DOCA)–salt hypertension, which is associated with a markedly depressed plasma renin activity because of sodium retention. However, the mechanisms underlying superoxide production in low-renin hypertension are undefined. Methods and Results—This study investigated (1) whether and how endothelin-1 (ET-1), which is increased in DOCA-salt hypertensive rats, contributes to arterial superoxide generation and (2) the effect of gene transfer of manganese superoxide dismutase and endothelial nitric oxide synthase. Both superoxide and ET-1 levels were significantly elevated in carotid arteries of DOCA-salt rats compared with that of the sham-operated controls. ET-1 concentration-dependently stimulated superoxide production in vitro in carotid arteries of normotensive rats. The increase in arterial superoxide in both ET-1–treated normotensive and DOCA-salt rats was reversed by a selective ETA receptor antagonist, ABT-627, the flavoprotein inhibitor diphenyleneiodonium, and the NADPH oxidase inhibitor apocynin but not by the nitric oxide synthase inhibitor N-L-arginine methyl ester or the xanthine oxidase inhibitor allopurinol. Furthermore, in vivo blockade of ETA receptors significantly reduced arterial superoxide levels, with a concomitant decrease of systolic blood pressure in DOCA-salt rats. Ex vivo gene transfer of manganese superoxide dismutase or endothelial nitric oxide synthase also suppressed superoxide levels in carotid arteries of DOCA-salt rats. Conclusions—These findings suggest that ET-1 augments vascular superoxide production at least in part via an ETA/NADPH oxidase pathway in low-renin mineralocorticoid hypertension. (Circulation. 2003;107:1053-1058.) Key Words: endothelin 䡲 NADPH oxidase 䡲 superoxide 䡲 hypertension
O
xidative stress and the inactivation of nitric oxide (NO) by vascular superoxide anion (O2⫺) play a critical role in the pathogenesis of vascular disease, including hypertension.1,2 Arterial O2⫺ is elevated in angiotensin II (Ang II)–induced hypertension,3 attributable to a large extent to NADPH oxidase activation by Ang II.4,5 However, an excess of vascular O2⫺ production has also been found in deoxycorticosterone acetate (DOCA)–salt hypertension,6 –9 a model with a markedly depressed plasma renin activity because of sodium retention.10 Humoral mechanisms responsible for O2⫺ production in mineralocorticoid hypertension remain to be delineated. In contrast to Ang II–induced hypertension, endothelin-1 (ET-1) has been shown to contribute to the pathogenesis of salt-sensitive hypertension in animals and humans,11 secondary to a low-renin state.12,13 ET-1 may be one of the most potent vasoconstrictors produced in the blood vessel wall to date.14 We have now found that the level of ET-1 is increased
in the arteries of DOCA-salt hypertensive rats. The vasoactive effects of ET-1 are mediated through 2 receptor types, ETA and ETB.15 ETA receptors play an important role in the development of DOCA-salt–induced hypertension, whereas ETB receptors may protect against vascular and renal injuries in this model.16 ET-1 is able to activate NADPH oxidase in endothelial cells17 and stimulates O2⫺ production in pulmonary smooth muscle cells.18 Therefore, we hypothesized that ET-1 activates NADPH oxidase to produce vascular O2⫺ in DOCA-salt hypertensive rats. Our results suggest that ET-1 produces O2⫺ via an ETA-NADPH oxidase pathway in carotid arteries of normotensive and DOCA-salt hypertensive rats. Because recent studies have suggested that endothelial NO synthase (eNOS) may also contribute to O2⫺ production when its essential cofactor BH4 is below the optimal level (ie, “uncoupled” eNOS),2 we used eNOS gene transfer in the present study in addition to NOS inhibition to distinguish the sources of O2⫺ generation. Gene transfer of manganese
Received September 26, 2002; revision received November 7, 2002; accepted November 8, 2002. From the Department of Pharmacology and Toxicology (L.L., G.D.F., S.W.W., C.A.N., J.J.G, A.F.C.) and the Neuroscience Program (G.D.F., J.J.G, A.F.C.), Michigan State University, East Lansing; and the Hypertension and Vascular Research Division (P.J.P.), Henry Ford Hospital, Detroit, Mich. Correspondence to Alex F. Chen, MD, PhD, FAHA, Department of Pharmacology and Toxicology, B403 Life Sciences Building, Michigan State University, East Lansing, MI 48824-1317. E-mail
[email protected] © 2003 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org
DOI: 10.1161/01.CIR.0000051459.74466.46
1053 Downloaded from http://circ.ahajournals.org/ by guest on March 16, 2015
1054
Circulation
February 25, 2003
superoxide dismutase (MnSOD) was also used to test the hypothesis that mitochondria may be a key source for O2⫺ formation. Our data indicate that local expression of these recombinant proteins significantly reduced vascular O2⫺ levels.
Methods DOCA-Salt Hypertensive Rats and In Vivo Pharmacological Intervention DOCA-salt hypertension was created in adult male Sprague-Dawley rats as previously described.9,19 Starting at week 3, some of the DOCA-salt rats received ABT-627 (Abbott Laboratories), a selective ETA receptor antagonist, 2 mg · kg body wt⫺1 · d⫺1 in drinking water for 2 weeks.20 Blood pressure was measured in conscious rats by the noninvasive tail-cuff method. The vessels were collected between weeks 4 and 5 after DOCA implantation. Animal procedures were in accordance with the institutional guidelines of the Michigan State University.
Ex Vivo Gene Transfer The preparation of adenoviral vectors was as described.9,21,22 Isolated arterial segments (4 mm) were transduced without (negative control) or with adenoviral vectors encoding eNOS, MnSOD, or -galactosidase (-gal) gene (positive control) at 5⫻1010 plaque formation units (pfu)/mL in minimal essential medium at 37°C for 4 hours, followed by incubation in fresh medium for 24 hours.9
Vascular O2ⴚ Levels
Vascular O2⫺ was assayed with oxidative dihydroethidium fluorescence and lucigenin (5 mol/L) chemiluminescence.9,23 To determine the effects of ET-1, ETA receptor, flavoprotein, NADPH oxidase, xanthine oxidase, and NOS-mediated O2⫺ production, arterial segments (4 mm) were preincubated at 37°C with ET-1 (0.01 to 1 nmol/L, 4 hours), ABT-627 (30 nmol/L, 60 minutes), diphenyleneiodonium (DPI, 0.1 mmol/L, 30 minutes), apocynin (0.1 mmol/L, 60 minutes), allopurinol (1 mol/L, 60 minutes), or N-L-arginine methyl ester (L-NAME, 0.1 mmol/L/L, 60 minutes), respectively. Arteries transduced with MnSOD, eNOS, and -gal (0 or 5⫻1010 pfu/mL) or collected after 2-week in vivo ABT-627 treatments were also assayed for O2⫺ levels.
ET-1 Immunoassay Vascular ET-1 levels were determined by a chemiluminescencebased immunoassay with a commercial kit (R&D Systems). Briefly, arteries from sham, DOCA, or normal rats treated with ET-1 were frozen in liquid nitrogen, homogenized in 1 mol/L acetic acid (1 mL/50 mg tissue) containing 1.5⫻10⫺5 mol/L pepstatin, and immediately boiled for 10 minutes. After being chilled, the homogenate was centrifuged at 20 000g for 30 minutes at 4°C, and the supernatant was assayed for ET-1 content.
Figure 1. Elevated ET-1 levels in carotid arteries of DOCA-salt rats. ET-1–treated carotid arteries of normal rats or carotid arteries of sham or DOCA-salt rats were subjected to ET-1 assay with a commercial enzyme immunoassay kit. n⫽4 to 6; **P⬍0.01 vs control, ##P⬍0.01 vs sham.
out ET-1 treatment. Arterial ET-1 content in DOCA-salt rats was comparable to that of arteries of normal rats treated with ET-1 for 4 hours at 10⫺9 mol/L (Figure 1, n⫽4 to 6, **P⬍0.01 versus control, ##P⬍0.01 versus sham).
Effect of ET-1 on O2ⴚ Production in Carotid Arteries of Normal Rats
Arterial O2⫺ levels of normal rats were increased in a concentration-dependent manner after incubation for 4 hours with ET-1, and pretreatment of ABT-627 (3⫻10⫺8 mol/L), a selective ETA receptor antagonist, completely reversed the effect of ET-1 on O2⫺ production (Figure 2, n⫽5 to 6, *P⬍0.05 and **P⬍0.01 versus control, #P⬍0.05 versus 10⫺9 mol/L ET-1 treated group).
In Vivo Blockade of ETA Receptors on Blood Pressure and Arterial O2ⴚ Levels in DOCA-Salt Rats There was a significant increase in average systolic blood pressure (Figure 3A, 176⫾4 versus 117⫾2 mm Hg, n⫽5, **P⬍0.01) and arterial O2⫺ levels (Figure 3B, *P⬍0.05 versus sham; see also Figure 6, E and F) in DOCA-salt rats compared with sham controls. In vivo blockade of ETA receptors for 2 weeks with ABT-627 significantly lowered blood pressure in DOCA-salt rats (Figure 3A, n⫽5, #P⬍0.05),
Data Analysis Data were expressed as mean⫾SEM. Repeated-measures ANOVA was used for comparison of multiple values obtained from the same subject, whereas factorial ANOVA was used for comparing data obtained from 2 independent samples of subjects. Bonferroni’s procedure was used to control type I error. A value of P⬍0.05 was considered significant.
Results ET-1 Levels in Carotid Arteries of DOCA-Salt Rats and Normal Rats After ET-1 Treatment ET-1 levels in carotid arteries were significantly higher in DOCA-salt rats than in sham controls. Similarly, ET-1 levels in arteries of normal rats treated with ET-1 for 4 hours were also significantly increased compared with the vessels with-
Figure 2. Effect of ET-1 on O2⫺ production in carotid arteries of normal rats. All vessel segments were incubated at 37°C for 4 hours without (control) or with ET-1 at increasing concentrations. ⫹ABT627 indicates that this group was incubated with ABT-627 (3⫻10⫺8 mol/L) for 1 hour before ET-1 treatment. All vessel segments were subjected to O2⫺ measurement by lucigenin-enhanced chemiluminescence assay (see Methods). n⫽5 to 6; *P⬍0.05 and **P⬍0.01 vs control, #P⬍0.05 vs 10⫺9mol/L ET-1–treated group.
Downloaded from http://circ.ahajournals.org/ by guest on March 16, 2015
Li et al
ET-1 and Superoxide in Low-Renin Hypertension
1055
Figure 5. Effect of gene transfer of MnSOD or eNOS on O2⫺ production in carotid arteries of DOCA-salt rats. Isolated carotid arteries of sham or DOCA-salt rats were transduced with adenoviral vectors encoding eNOS, MnSOD, or -gal for 4 hours at titers of 0 (control) and 5⫻1010 pfu/mL and then transferred to fresh medium overnight before O2⫺ assay. n⫽4 to 8; *P⬍0.05 vs DOCA, #P⬍0.05 vs sham.
Figure 3. A, Effect of ABT-627 (2 mg · g body wt⫺1 · d⫺1) on average systolic blood pressure in DOCA-salt rats (n⫽5; **P⬍0.01 vs sham rats, #P⬍ 0.05 vs DOCA-salt rats). B, Effect of ABT-627 (2 mg · kg body wt⫺1 · d⫺1) on O2⫺ production in carotid arteries of DOCA-salt rats. Isolated arterial segments of sham, DOCA-salt, or ABT-627–treated DOCA-salt rats were subjected to O2⫺ assay. n⫽5 to 6; *P⬍ 0.05 vs sham, #P⬍ 0.05 vs DOCA-salt group and P⬎0.05 vs sham.
with a concomitant decrease in arterial O2⫺ levels in the same group of DOCA-salt rats (Figure 3B, n⫽5, #P⬍ 0.05 versus DOCA).
Role of NADPH Oxidase, NOS, and Xanthine Oxidase on Arterial O2ⴚ Levels
DPI (10⫺4 mol/L), a flavoprotein inhibitor, and apocynin (10⫺4 mol/L), a selective NADPH oxidase inhibitor, significantly reduced arterial O2⫺ levels in DOCA-salt rats (Figure 4; see also Figure 6G). In contrast, allopurinol (10⫺6 mol/L) had no effect, and L-NAME (10⫺4 mol/L) increased arterial O2⫺ levels. Furthermore, both DPI and apocynin but not allopurinol significantly attenuated O2⫺ levels in arteries of normal rats treated with ET-1 (10⫺9 mol/L) (Figure 4, n⫽5 to 8, *P⬍0.05 and **P⬍0.01 versus control).
Gene Transfer of eNOS and MnSOD on O2ⴚ Levels in Carotid Arteries of DOCA-Salt Rats Arterial segments from sham or DOCA-salt rats were transduced with adenoviral vectors encoding eNOS, MnSOD, or -gal for 4 hours at titers of 0 (control) and 5⫻1010 pfu/mL and then transferred to fresh medium overnight before O2⫺ assay.9 Ex vivo gene transfer of either MnSOD or eNOS significantly decreased the arterial O2⫺ levels in DOCA-salt rats compared with the nontransduced controls of DOCA-salt rats that underwent the same medium incubation for 24 hours. In contrast, gene transfer of -gal had no effect on O2⫺ levels (Figure 5, n⫽4 to 8, *P⬍0.05 versus DOCA, #P⬍0.05 versus sham).
In Situ Detection of Vascular Superoxide In the presence of the superoxide-sensitive dye dihydroethidium, the ethidium bromide (EtBr) fluorescence (ie, red color) was markedly higher throughout the vessel wall of the ET-1–treated arteries of normal rat (Figure 6B) and arteries of DOCA-salt rats (Figure 6F) compared with the vessels from normal rats (Figure 6A) and sham rats (Figure 6E). The superoxide fluorescent intensity was dramatically suppressed in the arteries of DOCA-salt rats treated with DPI in vitro (Figure 6G) and arteries of DOCA-salt rats treated with ABT-627 for 2 weeks in vivo (Figure 6H) compared with the vessels from the control DOCA-salt rats (Figure 6F). Gene transfer of MnSOD (Figure 6I) and eNOS (Figure 6J) attenuated EtBr fluorescence in arteries of DOCA-salt rats. Both ABT-627 (Figure 6C) and DPI (Figure 6D) also suppressed the EtBr fluorescence in ET-1–treated arteries of normal rats.
Discussion Figure 4. Role of NADPH oxidase on O2⫺ production. Treatment with apocynin or DPI (10⫺4 mol/L) inhibited O2⫺ production in carotid arteries of DOCA-salt rats and in ET-1–treated (10⫺9 mol/L) carotid arteries of normal rats. Treatment with allopurinol (10⫺6 mol/L) had no significant effect on arterial O2⫺ levels, whereas treatment with L-NAME (10⫺4 mol/L) increased O2⫺ levels. n⫽5 to 8; *P⬍ 0.05 and **P⬍0.01 vs control.
The major new findings of this study are that ET-1 stimulates O2⫺ production, via an ETA/NADPH oxidase pathway, in carotid arteries of DOCA-salt hypertensive rats and that in vivo ETA receptor blockade attenuates systolic blood pressure and arterial O2⫺ levels. In addition, gene transfer of MnSOD or eNOS significantly reduces the increased arterial O2⫺ levels in this low-renin hypertension model.
Downloaded from http://circ.ahajournals.org/ by guest on March 16, 2015
1056
Circulation
February 25, 2003
Figure 6. Fluorescent confocal micrographs showing in situ O2⫺ detection in rat carotid arteries. Arterial sections were labeled with oxidative dye dihydroethidium, which fluoresces red when oxidized to ethidium bromide by superoxide (see Methods). A through D, Carotid arteries from normal rats: (A) control, (B) ET-1 treatment, (C) ABT-627 followed by ET-1 treatment, and (D) DPI followed by ET-1 treatment. E through J, Carotid arteries of DOCA-salt or sham rats: (E) sham, (F) DOCA-salt, (G) in vitro DPI-treated DOCA-salt, (H) in vivo ABT-627–treated DOCA-salt, (I) MnSOD gene–transferred DOCA-salt, and (J) eNOS gene– transferred DOCA-salt. Sections shown are typical of 3 separate experiments. Bar⫽0.05 mm.
Arterial superoxide levels are markedly increased in DOCA-salt hypertensive rats.6 –9 However, it was not clear which factors are responsible for the augmented superoxide production. It is well known that ET-1 plays an important role in DOCA-salt hypertension. ET-1 is implicated in the development and maintenance of hypertension at least in part because of its potent vasoconstrictor property.11–13 Our results demonstrate that there was a much higher ET-1 level in carotid arteries of DOCA-salt rats than in sham controls, an observation that is consistent with published studies showing enhanced vascular mRNA expression and ET-1 contents in the resistance arteries of this hypertension model.14 Furthermore, our data suggest that arterial ET-1 levels in DOCA-salt hypertensive rats were comparable to those observed in the normal arteries treated with 10⫺9 mol/L of ET-1 from normal rats.
It was recently reported that ET-1 activates NADPH oxidase and induces superoxide production in cultured endothelial and smooth muscle cells.17,18 Convincing evidence indicates that the major enzymatic sources for vascular superoxide formation are NADPH oxidase, xanthine oxidase, and uncoupled NOS.2 In DOCA-salt hypertensive rats, aortic NADPH oxidase activity was significantly increased compared with their normotensive controls.7,8 In the present study, we examined (1) the effect of ET-1 on superoxide production both in vitro in normal rats and in vivo in DOCA-salt hypertensive rats and (2) whether this effect is mediated by NADPH oxidase, xanthine oxidase, or uncoupled NOS. Our results indicate that (1) ET-1 stimulates arterial O2⫺ production in a concentration-dependent manner in normal rats; (2) apocynin but not L-NAME or allopurinol inhibits the O2⫺ production in both ET-1–stimulated arteries of normal rats and arteries of DOCA-salt rats; and (3) the selective ETA receptor antagonist ABT-627 suppresses superoxide production in vitro in ET-1–treated arteries of normal rats and in vivo in arteries of DOCA-salt hypertensive rats. Collectively, these data suggest that ET-1 stimulates arterial O2⫺ production in DOCA-salt hypertension, and NADPH oxidase but not xanthine oxidase or uncoupled NOS may play a major role in O2⫺ production in this model. The selectivity of apocynin, a methoxy-substituted catechol, on NADPH oxidase has been well characterized, because it impedes the assembly of the p47phox and p67phox subunits within the membrane NADPH oxidase complex.8,24 There are at least 2 vascular ET-1 receptors, ETA and ETB.15 ET-1 exerts its vasoactive effects mainly through the activation of the G protein– coupled ETA receptors on vascular smooth muscle cells,16 whereas ETB may exert protective effects in DOCA-salt hypertension.16,25 There is an exaggerated vascular and renal injury in ETB receptor– deficient rats of DOCA-salt hypertension, and such injuries were significantly improved after the treatment with ABT-627, a selective ETA receptor antagonist.25 In the present study, we demonstrated that arterial O2⫺ levels were increased significantly in DOCA-salt rats compared with the sham controls, an effect that was reversed after in vivo ABT-627 treatment in DOCA-salt rats, with a concomitant reduction of blood pressure. These findings are consistent with recent studies showing that O2⫺ production in rebound pulmonary hypertension was mediated by ETA receptors18 and that tempol, a superoxide scavenger, normalized blood pressure in spontaneously hypertensive rats.26 However, it is important to note that although in vivo blockade of ETA receptors by ABT-627 for 2 weeks suppressed the arterial O2⫺ to control levels, the blood pressure was only partially reduced. These results suggest that ETA receptor–mediated activation of NADPH oxidase by ET-1 is only one of the contributing factors for the O2⫺-induced blood pressure increase in this model of hypertension. In addition, they also suggest that the reduced vascular O2⫺ levels were only partially responsible for decreasing blood pressure and that in vivo ETA receptor blockade may also result in reduced smooth muscle tension. Furthermore, the possible influence of ETB receptors on ET-1–induced O2⫺ production in arteries and veins is not clear and is a subject currently being investigated. Finally, it
Downloaded from http://circ.ahajournals.org/ by guest on March 16, 2015
Li et al is also of interest to note that hypertension per se may not be a major stimulus for augmented vascular superoxide because norepinephrine-induced hypertension is not associated with an increase in vascular superoxide levels.4 Thus, O2⫺ production may be a result of the effects of different vasoactive agents in different types of hypertension. Although Ang II is clearly a key stimulus of vascular O2⫺ production in high-angiotensin hypertension, ET-1 may play a major role for increasing vascular O2⫺ in low-renin hypertension, such as the DOCA-salt model. Several pathophysiological conditions in addition to hypertension have been associated with increased superoxide production. These include atherosclerosis, hypercholesterolemia, diabetes, and heart failure; cigarette smoking has also been implicated.4 In most of these cases, the increase in vascular O2⫺ has been shown to impair endotheliumdependent NO-mediated vascular relaxation by inactivating endogenous NO.4 In addition, superoxide has also been shown to affect the sensitivity of blood vessels to vasodilators,27 and it triggers expression of vascular adhesion molecules and vascular remodeling.9 The reaction rate between O2⫺ and NO is linear and extremely rapid.28 For this reason, the local balance between O2⫺, NO, and SOD in the vascular wall is dynamic, and relatively minor changes in the levels of any of these factors may substantially alter vascular tone. Localized gene transfer to the vessel wall may be an effective means of increasing NO and/or reducing O2⫺ levels. Indeed, our results demonstrate that gene transfer of MnSOD or eNOS significantly reduced arterial O2⫺ levels in DOCA-salt hypertensive rats. We chose MnSOD for vascular gene transfer because (1) our recent study indicates that endogenous MnSOD is significantly reduced in the carotid arteries of DOCA-salt hypertensive rats and that gene transfer of MnSOD restored the functional capacity of the antioxidant enzyme in scavenging elevated O2⫺9 and (2) mitochondria may be a major location in which vascular O2⫺ is produced.29 In agreement with our findings, gene transfer of MnSOD has been shown to normalize superoxide-induced impairment of endothelium-dependent relaxation,30,31 whereas gene transfer of cytosolic Cu/Zn-SOD or extracellular SOD did not.32–34 Conversely, recent studies have demonstrated that both ex vivo and in vivo gene transfer of eNOS or nNOS restored NO-mediated arterial relaxation, which was impaired by increased O2⫺ in hypertensive,34,35 atherosclerotic,31,33,36 –38 or diabetic animals.30,39 Furthermore, in vivo gene transfer of eNOS to spontaneously hypertensive rats has resulted in direct blood pressure reduction.40 Consistent with these studies, our data showed that gene transfer of eNOS significantly decreased O2⫺ levels in carotid arteries in DOCA-salt rats. Taken together, these experimental observations support the novel concept that NO generated by recombinant NOS, as a result of vascular gene transfer, provides an effective means of inactivating O2⫺ and thereby improving vasomotor function. In conclusion, the present study demonstrates that ET-1 is a potent stimulus for arterial O2⫺ produced in low-renin DOCA-salt hypertension, an effect that is at least partially mediated by the ETA receptor/NADPH oxidase pathway. Vascular gene transfer of MnSOD and eNOS is an effective
ET-1 and Superoxide in Low-Renin Hypertension
1057
strategy in reducing O2⫺ levels in this model. These findings may provide a mechanistic basis for therapeutic interventions aimed at reducing superoxide-induced vascular dysfunctions associated with increased ET-1 levels in low-renin hypertension.
Acknowledgments This work was supported in part by the American Heart Association, grants 9806347X and 0130537Z; the American Diabetes Association, research award 7-01-RA-10; and the Juvenile Diabetes Research Foundation, innovative grant 5-2001-311 (to Dr Chen). Dr Li is an awardee of the AHA/Midwest Affiliate Physician-Scientist Postdoctoral Fellowship (0225408Z).
References 1. Griendling KK, Alexander RW. Oxidative stress and cardiovascular disease. Circulation. 1997;96:3264 –3265. 2. Landmesser U, Harrison DG. Oxidant stress as a marker for cardiovascular events: ox marks the spot. Circulation. 2001;104:2638 –2640. 3. Oskarsson HJ, Heistad DD. Oxidative stress produced by angiotensin II: implications for hypertension and vascular injury. Circulation. 1997;95: 557–559. 4. Rajagopalan S, Curz S, Munzel T, et al. Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation: contribution to alterations of vasomotor tone. J Clin Invest. 1996;97:1916 –1923. 5. Pagano PJ, Clark JK, Cifuentes-Pagano ME, et al. Localization of a constitutively active, phagocyte-like NADPH oxidase in rabbit aortic adventitia: enhancement by angiotensin II. Proc Natl Acad Sci U S A. 1997;94:14483–14488. 6. Somers MJ, Mavromatis K, Galis ZS, et al. Vascular superoxide production and vasomotor function in hypertension induced by deoxycorticosterone acetate-salt. Circulation. 2000;101:1722–1728. 7. Wu R, Millette E, Wu L, et al. Enhanced superoxide anion formation in vascular tissues from spontaneously hypertensive and deoxycorticosterone acetate-salt hypertensive rats. J Hypertens. 2001;19:741–748. 8. Beswick RA, Dorrance AM, Leite R, et al. NADH/NADPH oxidase and enhanced superoxide production in the mineralocorticoid hypertensive rat. Hypertension. 2001;38:1107–1111. 9. Li LX, Crockett E, Wang DH, et al. Gene transfer of endothelial NO synthase and manganese superoxide dismutase on arterial vascular cell adhesion molecule-1 expression and superoxide production in deoxycorticosterone acetate-salt hypertension. Arterioscler Thromb Vasc Biol. 2002;22:249 –255. 10. Gavras H, Brunner HR, Laragh JH, et al. Malignant hypertension resulting from deoxycorticosterone acetate and salt excess: role of renin and sodium in vascular changes. Circ Res. 1975;36:300 –309. 11. Schiffrin EL. Role of endothelin-1 in hypertension and vascular disease. Am J Hypertens. 2001;14:83S– 89S. 12. Letizia C, Cerci S, De Toma G, et al. High plasma endothelin-1 levels in hypertensive patients with low-renin essential hypertension. J Hum Hypertens. 1997;117:447– 451. 13. Elijovich F, Laffer CL, Amador E, et al. Regulation of plasma endothelin by salt in salt-sensitive hypertension. Circulation. 2001;103:263–268. 14. Lariviëre R, Thibault G, Schiffrin EL. Increased endothelin-1 content in blood vessels of deoxycorticosterone acetate-salt hypertensive but not in spontaneously hypertensive rats. Hypertension. 1993;21:294 –300. 15. Luscher TF, Barton M. Endothelins and endothelin receptor antagonists: therapeutic considerations for a novel class of cardiovascular drugs. Circulation. 2000;102:2434 –2440. 16. Matsumura Y, Hashimoto N, Taira S, et al. Different contribution of endothelin-A and endothelin-B receptors in the pathogenesis of deoxycorticosterone acetate-salt–induced hypertension in rats. Hypertension. 1999;33:759 –765. 17. Duerrschmidt N, Wippich N, Goettsch W, et al. Endothelin-1 induces NAD(P)H oxidase in human endothelial cells. Biochem Biophys Res Commun. 2000;269:713–717. 18. Wedgwood S, McMullan M, Bekker JM, et al. Role for endothelin1–induced superoxide and peroxynitrite production in rebound pulmonary hypertension associated with inhaled nitric oxide therapy. Circ Res. 2001;89:357–364.
Downloaded from http://circ.ahajournals.org/ by guest on March 16, 2015
1058
Circulation
February 25, 2003
19. Watts SW, Fink GD. 5-HT2B-receptor antagonist LY-272015 is antihypertensive in DOCA-salt-hypertensive rats. Am J Physiol. 1999;276: H944 –H952. 20. Ballew JR, Fink GD. Role of ETA receptors in experimental Ang II-induced hypertension in rats. Am J Physiol. 2001;281:R150 –R154. 21. Chen AF, O’Brien T, Tsutsui M, et al. Expression and function of recombinant endothelial nitric oxide synthase gene in canine basilar artery. Circ Res. 1997;80:327–335. 22. Chen AF, Jiang S, Crotty TB, et al. Effects of in vivo adventitial expression of recombinant endothelial nitric oxide synthase gene in cerebral arteries. Proc Natl Acad Sci U S A. 1997;94:12568 –12573. 23. Cifuentes ME, Rey FE, Carretero OA, et al. Upregulation of p67phox and gp91phox in aortas from angiotensin II-infused mice. Am J Physiol. 2000; 279:H2234 –H2240. 24. Meyer JW, Schmitt ME. A central role for the endothelial NADPH oxidase in atherosclerosis. FEBS Lett. 2000;472:1– 4. 25. Matsumura Y, Kuro T, Kobayashi Y, et al. Exaggerated vascular and renal pathology in endothelin-B receptor-deficient rats with deoxycorticosterone acetate-salt hypertension. Circulation. 2000;102: 2765–2773. 26. Schnackenberg CG, Welch WJ, Wilcox CS. Normalization of blood pressure and renal vascular resistance in SHR with a membranepermeable superoxide dismutase mimetic: role of nitric oxide. Hypertension. 1998;32:59 – 64. 27. Wu L, de Champlain J. Effects of superoxide on signaling pathways in smooth muscle cells from rats. Hypertension. 1999;34:1247–1253. 28. Thomson L, Trujillo M, Telleri R, et al. Kinetics of cytochrome oxidation by peroxynitrite: implications for superoxide measurements in nitric oxide-producing biological systems. Arch Biochem Biophys. 1995;319: 491– 497. 29. McIntyre M, Bohr DF, Dominiczak AF. Endothelium function in hypertension: the role of superoxide anion. Hypertension. 1999;34:539 –545.
30. Zanetti M, Sato J, Katusic ZS, et al. Gene transfer of superoxide dismutase isoforms reverses endothelial dysfunction in diabetic rabbit aorta. Am J Physiol. 2001;280:H2516 –H2523. 31. Zanetti M, Sato J, Jost CJ, et al. Gene transfer of manganese superoxide dismutase reverses vascular dysfunction in the absence but not in the presence of atherosclerotic plaque. Hum Gene Ther. 2001;12:1407–1416. 32. Miller FJ, Gutterman DD, Rios CD, et al. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res. 1998;82:1298 –1305. 33. Mozes G, Kullo IJ, Mohacsi TG, et al. Ex vivo gene transfer of endothelial nitric oxide synthase to atherosclerotic rabbit aortic rings improves relaxations to acetylcholine. Atherosclerosis. 1998;141:265–271. 34. Nakane H, Miller FJ Jr, Faraci FM, et al. Gene transfer of endothelial nitric oxide synthase reduces angiotensin II–induced endothelial dysfunction. Hypertension. 2000;35:595– 601. 35. Alexander MY, Brosnan MJ, Hamilton CA, et al. Gene transfer of endothelial nitric oxide synthase but not Cu/Zn superoxide dismutase restores nitric oxide availability in the SHRSP. Cardiovasc Res. 2000; 47:609 – 617. 36. Ooboshi H, Toyoda K, Faraci FM, et al. Improvement of relaxation in an atherosclerotic artery by gene transfer of endothelial nitric oxide synthase. Arterioscler Thromb Vasc Biol. 1998;18:1752–1756. 37. Channon KM, Qian H, Neplioueva V, et al. In vivo gene transfer of nitric oxide synthase enhances vasomotor function in carotid arteries from normal and cholesterol-fed rabbits. Circulation. 1998;98:1905–1911. 38. Sato J, Mohacsi T, Noel A, et al. In vivo gene transfer of endothelial nitric oxide synthase to carotid arteries from hypercholesterolemic rabbits enhances endothelium-dependent relaxations. Stroke. 2000;31:968 –975. 39. Lund DD, Faraci FM, Miller FJ Jr, et al. Gene transfer of endothelial nitric oxide synthase improves relaxation of carotid arteries from diabetic rabbits. Circulation. 2000;101:1027–1033. 40. Lin KF, Chao L, Chao J. Prolonged reduction of high blood pressure with human nitric oxide synthase gene delivery. Hypertension. 1997;30:307–313.
Downloaded from http://circ.ahajournals.org/ by guest on March 16, 2015
Endothelin-1 Increases Vascular Superoxide via EndothelinA−NADPH Oxidase Pathway in Low-Renin Hypertension Lixin Li, Gregory D. Fink, Stephanie W. Watts, Carrie A. Northcott, James J. Galligan, Patrick J. Pagano and Alex F. Chen Circulation. 2003;107:1053-1058; originally published online February 3, 2003; doi: 10.1161/01.CIR.0000051459.74466.46 Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2003 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circ.ahajournals.org/content/107/7/1053
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation is online at: http://circ.ahajournals.org//subscriptions/
Downloaded from http://circ.ahajournals.org/ by guest on March 16, 2015
