IMPAIRED ENDOTHELIAL FUNCTION AND MICROVASCULAR ADMA IN ANGIOTENSIN II INFUSED RATS: EFFECTS OF TEMPOL

Abstract

Angiotensin II causes endothelial dysfunction which is associated with cardiovascular risk. We investigated the hypothesis that angiotensin II increases microvascular reactive oxygen species and asymmetric dimethylarginine and switches endothelial function from vasodilator to vasoconstrictor pathways. Acetylcholine-induced endothelium dependent responses of mesenteric resistance arterioles were assessed in a myograph and vascular nitric oxide and reactive oxygen species by fluorescent probes in groups (n=6) of male rats infused for 14 days with angiotensin II (200 ng/kg/min) or given a sham infusion. Additional groups of angiotensin or sham-infused rats were given oral tempol (2 mmol · l⁻¹). Angiotensin II infusion increased mean blood pressure (119±5 vs 89±7 mmHg; P<0.005) and plasma malondialdehyde (0.57±0.02 vs 0.37±0.05μmol · l⁻¹; P<0.035) and decreased maximal endothelium dependent relaxation (18±5 vs 54±6%, P<0.005) and hyperpolarizing (19±3 vs 29±3%, P<0.05) responses and nitric oxide activity (0.9±0.1 vs 1.6±0.2 Units, P<0.01) yet enhanced endothelium dependent contraction responses (23±5 vs 5±5%, P<0.05) and reactive oxygen species production (0.82±0.05 vs 0.15±0.03 units; P<0.01). Angiotensin II decreased the expression of dimethylarginine dimethylaminohydrolase-2 and increased asymmetric dimethylarginine in vessels (450±50 vs 260±35 pmol/mg protein, P<0.01) but not plasma. Tempol prevented any significant changes with angiotensin II. In conclusion, angiotensin redirected endothelial responses from relaxation to contraction, reduced vascular nitric oxide and increased asymmetric dimethylarginine. These effects were dependent on reactive oxygen species and could, therefore, be targeted with effective antioxidant therapy.

Introduction

Normal vessels display endothelium-dependent relaxation responses to acetylcholine (Ach). The endothelium-dependent relaxation factor (EDRF) is mediated by endothelial nitric oxide synthase (NOS-3). The mediators of the endothelium-dependent hyperpolarizing factor (EDHF) vary by species and vascular bed, but can include hydrogen peroxide (H₂O₂) ¹, epoxyeicosatrienoic acids (EETs) ² and electro-mechanical coupling ³. These cause activation of calcium-dependent potassium channels (K_Ca) on VSMCs that can be blocked by a combination of apamin and charybdotoxin ². Some vessels also display a cyclooxygenase (COX)-dependent relaxation response attributable to prostacyclin (PGI₂) although this was not apparent in rat afferent arterioles ². An endothelium-dependent contracting factor (EDCF) occurs in models with increased reactive oxygen species (ROS). The EDCF in renal afferent arterioles from rabbits infused with Ang II entailed a vasoconstrictor prostaglandin generated by COX that activated thromboxane prostanoid receptors (TP-Rs) on VSMCs ⁴.

Ang II generates ROS in blood vessels that can bioinactivate NO ⁴. However, NOS-dependent EDRF responses of resistance arterioles are not consistently reduced ^4,5 by angiotensin infusion. Moreover, ROS can generate an EDCF ^4,6. However, it is not clear whether prolonged inhibition of ROS can prevent the defects in endothelium-dependent relaxations and prevent the EDCF responses of resistance vessels from animals infused with Ang II.

Increasing evidence suggests that an endogenous inhibitor of nitric oxide synthase (NOS), asymmetric dimethylarginine (ADMA), and its metabolism by dimethylarginine dimethylaminohydrolase (DDAH) ⁷ may regulate vascular NO. Angiotensin II infusion can increase circulating levels of ADMA ^8,9. However, it is unclear whether increased ADMA levels in plasma or tissues can be prevented by an effective antioxidant. We recently reported that culture of VSMCs with Ang II increased cellular, but not medium, levels of ADMA ¹⁰. To resolve these issues, we assessed the three major endothelium-dependent pathways and related these responses to vascular NO and ROS activities and plasma and tissue ADMA concentrations in mesenteric resistance arteries in rats during a slow pressor infusion of angiotensin II. Other groups of sham and angiotensin II-infused rats received the antioxidant drug tempol throughout ¹¹.

These experiments were designed to test the hypothesis that ROS, generated in microvascular resistance vessels during prolonged Ang II-induced hypertension, impairs EDRF, EDHF and NO activity and increases microvascular ADMA and EDCF. We selected tempol, which is a redox cycling nitroxide, to reduce ROS and enhance vascular NO ¹¹. These experiments are significant because restoration of endothelial function and NO, and abrogation of EDCF, ROS and ADMA, would reverse some of the earliest manifestations of hypertension and vascular disease ¹².

Methods

Male Sprague Dawley rats (200–220; Taconics Lab, Germantown, NY) were maintained on tap water and standard chow (Na⁺ 0.4g · 100g⁻¹) under constant humidity and temperature and 12 hour light-dark cycles. The protocols were approved by the Institutional Animal Care and Use Committee of Georgetown University. Details of methods appear in supplement (Please see http://hyper.ahajournals.org)

Animal model

Three sets each of 4 groups of Sprague-Dawley rats were prepared (6 per group were used). Rats were anesthetized with sodium pentobarbital (50 mg · kg⁻¹) for subcutaneous insertion of a sham mini pump (sham) or an osmotic mini pump (model 202; Alzato Palo Alto CA) at the dorsum of the neck. Group 1 had a sham infusion. Group 2 received Ang II (Peninsula Lab, San Carlos, CA) at 200ng · kg⁻¹ · min⁻¹ for 14 days. Group 3 received a sham mini pump and oral tempol (2 mmol · l⁻¹ of water). Group 4 received Ang II mini pump and tempol. On the experimental day, rats were anesthetized with inhaled isoflurane (3% balance with oxygen). The carotid artery was cannulated for measurement of mean arterial pressure (MAP; model DPM-1B, Biotek, VT, USA). Blood was withdrawn and the plasma was stored at −80°C. Thereafter, rats were euthanized by exsanguination, the abdomen was opened and mesenteric arteries were isolated and studied in a myograph or snap frozen and stored at −80°C for analysis of ADMA or protein.

EDRF/NO, EDHF and EDCF responses of mesenteric resistance arterioles

The mesenteric vessels were isolated, mounted in a Mulvany-Halpern myograft (JP Trading, Science Park Aarhus, Denmark) and studied as described in detail previously ¹³, described in the supplement and summarized in Figure S1 (see supplement).

Fluorescence detection of acetylcholine–induced nitric oxide and ROS in mesenteric resistance arterioles

These followed methods ¹³ described in details in the supplement.

Protein expression studies

The protein expression in lysates of isolated mesenteric resistance arterioles were studied as described ¹³.

Plasma malondialdehyde

Plasma malondialdehyde was measured by High Performance Capillary Electrophoresis-Micellar Electrokinetic Chromatography (see supplement).

L-arginine, asymmetric - and symmetric - dimethylarginine concentrations in plasma and tissue

For details, see supplement to page 3.

Statistics

Values are expressed as mean ± SEM. The concentration-response curves to Ach were analyzed using nonlinear regression of sigmoidal concentration-response curves (GraphPad Prism), which were used to calculate the EC₅₀. Other values were analyzed by ANOVA followed by a post hoc test for multiple comparisons (GraphPad Prism). A value of P<0.05 was considered statistically significant.

Results

Mean arterial pressure under anesthesia and plasma malondialdehyde were increased by Ang II, but this was prevented in rats receiving tempol. There were no changes in plasma arginine, ADMA or SDMA (Table 1).

Table 1.

Effects of angiotensin II infusion alone or with tempol on mean arterial pressure and plasma malondialdehyde, L-arginine, ADMA and SDMA

Variable	Sham	Ang II	Tempol	Tempol + Ang II
MAP (mmHg)	89 ± 7	119 ± 5†	90.0 ± 3	98.2 ± 6
Malondialdehyde (μmol · l−1)	0.37 ± 0.05	0.57 ± 0.02*	0.46 ± 0.05	0.45 ± 0.05
L-arginine (μmol · l−1)	173 ± 13	179 ± 55	137 ± 48	141 ± 66
ADMA (nmol · l−1)	510 ± 46	594 ± 127	610 ± 46	700 ± 20
SDMA (nmol · l−1)	280 ± 30	309 ± 31	300 ± 12	300 ± 9

Mean ± SEM values (n=6 per group). Effects of Ang II:

^*P<0.05;

^†P<0.005.

Figure S1 depicts how endothelium-dependent responses were assessed (see Fig S1 in supplement). Responses to Ach are shown in Figure 1 and EC₅₀ and maximal values in Table 2. Compared to sham-infused rats, those infused with Ang II had a 57% reduction in maximal relaxation with Ach, a 67% reduction in EDRF but a more modest 34% reduction in EDRF. Ang II enhanced EDCF responses by 4 fold. Ang II infusion also reduced the sensitivity of the Ach and EDRF responses but increased the sensitivity of the EDCF responses and did not change the sensitivity of EDHF responses. There were no significant effects of Ang II infusion on these responses in rats administered tempol (Table 2).

Figure 1.

Mean ± SEM values for relaxation responses to acetylcholine in norepinephrine preconstricted mesenteric arterioles (panel A) and calculated EDRF (panel B) and EDHF (panel C) and EDCF responses in vessels under spontaneous tone (panel D). Comparing sham- and angiotensin-infused responses: *, P<0.05; **, P<0.01. There were no significant differences between sham- and angiotensin-infused responses in rats given tempol. B, before.

Table 2.

Maximal relaxation responses and EC₅₀ values of vessels incubated with acetylcholine to Ach of mesenteric arteries from vehicle, Ang II, tempol, tempo with Ang II infusion rats

Response	Maximum (%)	Maximum (%)	P	Log EC50 (mol/L)	Log EC50 (mol/L)	P
				Vehicle	Ang II
Ach	83.9 ± 7.9	36.0 ± 7.0	<0.01	−6.17 ± 0.06	−5.47 ± 0.04	<0.005
EDRF	54.4 ± 4.6	18.0 ± 4.7	<0.01	−6.55 ± 0.06	−5.78 ± 0.05	<0.005
EDHF	29.4 ± 2.7	18.7 ± 2.7	<0.05	−5.18 ± 0.07	−5.46 ± 0.16	ns
EDCF	5.2 ± 4.6	23.0 ± 5.6	<0.05	−4.11 ± 0.35	−4.49 ± 0.03	<0.005
				Tempol	Tempol-Ang II
Ach	88.9 ± 7.9	69.9 ± 8.9	ns	−6.21 ± 0.05	−6.16 ± 0.08	ns
EDRF	53.4 ± 5.7	41.4 ± 4.7	ns	−6.76 ± 0.09	−6.67 ± 0.01	ns
EDHF	35.4 ± 3.7	27.4 ± 4.7	ns	−5.38 ± 0.15	−5.15 ± 0.14	ns
EDCF	5.1 ± 6.2	10.4 ± 4.6	ns	−4.16 ± 0.13	−4.44 ± 0.07	ns

Data were obtained from experiments described in Figure 1.

Further data for mesenteric resistance arterioles are shown in Table 3. Ach-induced DAFFM-NO fluorescence was reduced by 41% in vessels from Ang-infused rats, whereas acetylcholine-induced Tempo-9AC-ROS fluorescence was increased five-fold (Table 3). Both of these changes were prevented in rats given tempol. Tissue concentrations of L-arginine and SDMA in mesenteric vessels were unaffected by Ang II infusion, but ADMA was increased significantly by 73%. This was prevented by tempol, which led to a significant increase in tissue L-arginine in rats infused with Ang II. The expression of DDAH-2 protein was reduced by Ang II infusion but was prevented in rats given tempol.

Table 3.

Effects of angiotensin II infusion alone or during tempol administration on mesenteric resistance arterioles

Variable	Sham	Ang II	Tempol	Tempol + Ang II
NO activity with Ach (F/Fo DAF)	1.6 ± 0.2	1.0 ± 0.1‡	1.7 ± 0.2	1.3 ± 0.1
ROS activity with Ach (F/Fo tempo 9AC)	0.15 ± 0.03	0.82 ± 0.05‡	0.22 ± 0.05	0.42 ± 0.11
L-arginine (nmol · mg protein −1)	71 ± 25	95 ± 23	33 ± 4	57 ± 8*
ADMA (pmol · mg protein −1)	260 ± 35	450 ± 50†	228 ± 36	312 ± 36
SDMA (pmol · mg protein −1)	80 ± 50	127 ± 76	56 ± 9	103 ± 28
DDAH-2 protein (relative to β-actin)	1.4 ± 0.2	0.7 ± 0.1‡	1.2 ± 0.3	1.1 ± 0.2

Mean ± SEM values (n=6 per group) for mesenteric resistance arterioles. Effects of angiotensin II:

^*P<0.05;

^†P<0.01;

^‡P<0.005.

We evaluated whether a reduced response to NO, H₂O₂ or EETs could account for the reduced EDRF or EDHF responses of vessels from Ang-infused rats. The maximal relaxation to the NO donor, sodium nitroprusside in vessels from sham-infused rats (10⁻⁴ M; 97 ± 8%) was not significantly different (94 ± 6%) in vessels from Ang II-infused rats. H₂O₂ elicited a modest relaxation of 9 ± 3% at 10⁻⁵ mol · l⁻¹ and a marked relaxation at 10⁻⁴ mol · l⁻¹. However, these responses were similar in vessels from sham- and Ang-infused rats (Fig S2A in supplement). EETs did not elicit a significant relaxation even at 10⁻⁵ mol · l⁻¹ (Fig S2B in supplement). Therefore, differences in responsiveness to NO, H₂O₂ or EETs could not explain the reduced relaxation responses from Ang-infused rats.

To assess the role of vasodilator prostaglandins (PGs) in EDRF responses, vessels from sham-infused rats were incubated with 5 × 10⁻⁵ mol · l⁻¹ of indomethacin (Indo; to block COX-1 and -2). Relaxation responses to 10⁻⁴ M acetylcholine after vehicle (84 ± 8%) were unchanged after inhibition of cyclooxygenase with 5 × 10⁻⁵ mol · l⁻¹ of Indo (79 ± 6%) but were abolished by endothelium removal (1 ± 3%). Therefore, Indo was not used in subsequent relaxation studies.

The mediation of EDHF was studied in vessels from sham-infused rats with the use of specific inhibitors (Table 4A). EDHF response to 10⁻⁴ M acetylcholine with vehicle was 29 ± 3%. This was not significantly blunted by catalase to metabolize H₂O₂ or by 14,15-epoxyeicosa-5-(Z)-enoic acid (14,15EEZE) to block EETs ¹⁴ in concentrations shown to be effective in isolated vessels ^2,15 but was abolished by endothelium removal. Thus, the EDHF response depended on the endothelium but were not mediated significantly by H₂O₂ or EETs.

Table 4A.

Maximal endothelium-dependent hyperpolarization responses in mesenteric resistance arterioles from sham-infused rats

Added to the bath	Relaxation (%)	P value vs vehicle
Vehicle	29 ± 3
Catalase (125 units · ml−1)	26 ± 6	ns
14,15-EEZE (10−5M)	32 ± 5	ns
Endothelium removal	1 ± 5	P<0.005

Mean ± SEM values (n=6 per group). All vessels were preconstricted with 10⁻⁵ mol · l⁻¹ norepinephrine, pretreated with 10⁻⁴ mol · l⁻¹ l-nitroarginine to inhibit nitric oxide synthase and relaxed with 10⁻⁴ mol · l⁻¹ acetylcholine. ns = not significant

The EDCF response of vessels from Ang-infused rats (Table 4B) was not reduced significantly by incubation with parecoxib to block COX-2 or with OKY-046 to block thromboxane synthase. However, the response was inhibited similarly by SC-560 to block COX-1 or SQ-29,548 to block thromboxane prostanoid receptors (TP-Rs) and was abolished by endothelium removal. These drug concentrations were fully effective in isolated vessels ⁴. Thus, EDCF responses depended on the endothelium and COX-1 products that activated TP-Rs.

Table 4B.

Maximum endothelium-dependent contraction factor responses in mesenteric resistance arterioles from angiotensin II-infused rats

Added to bath	Contraction (%)	P value vs vehicle
Vehicle	23 ± 5
SC-560 (10−7M)	7 ± 5	P<0.005
Parecoxib (10−5M)	19 ± 4	ns
OKY-046 NA (10−5M)	13 ± 3	ns
SQ-29,584 (10−5M)	6 ± 4	P<0.005
Endothelium removal	1 ± 4	P<0.005

Mean ± SEM values (n=6 per group). Vessels were studied under spontaneous tone. They were pretreated with 10⁻⁴ mol · l⁻¹ l-nitroarginine, 10⁻⁶ mol · l⁻¹ apamin and 10⁻⁵ mol · l⁻¹ charybdotoxin to block relaxation responses and constricted with 10⁻⁴ mol · l⁻¹ acetylcholine. ns = not significant

Concentrations of L-arginine, ADMA and SDMA in homogenates of aorta, kidney and liver are shown in Table S1 in supplement. Tissue levels of L-arginine and SDMA were unaffected by Ang II infusion but levels of ADMA in all three tissues were increased. These effects were prevented in rats given tempol.

Discussion

The main new findings are that a prolonged slow pressor infusion of Ang II into rats changed endothelial function of mesenteric resistance vessels by diminishing vasodilator and increasing vasoconstrictor pathways. This was accompanied by a reduced endothelial release of microvascular NO, and increased ROS and ADMA. Angiotensin infusion downregulated the vascular expression of DDAH-2. These effects of Ang II were prevented by two weeks of oral tempol administration. Plasma ADMA did not predict vascular, renal or hepatic tissue levels of ADMA.

We confirmed previous findings that oral tempol prevented the increased blood pressure with a slow pressor infusion of Ang II ^11,16. Vascular ROS with Ang II are derived from NADPH oxidase activity ¹⁷ and a reduction in extracellular superoxide dismutase expression ¹⁸. Tempol prevented superoxide anion (O₂^·−) generation by Ang II in VSMCs ¹⁹ and inhibited lipid peroxidation and microvascular ROS in response to Ang II in the present study. We selected a slow pressor rate of Ang II infusion in this study since high rates of Ang II infusion caused renal damage that reduced DDAH activity ⁸.

A reduction in EDRF in microvessels of rabbits infused with Ang II was improved by bath addition of tempol ²⁰. A reduced EDRF has been related to bioinactivation of NO by ROS ¹¹. This is consistent with our findings that tempol administration prevented the reduction in microvascular EDRF and NO, and the increase in ROS, in rats infused with Ang II.

The maximal EDHF response was also reduced by prolonged Ang II infusion but the effect was smaller than on EDRF and the EC₅₀ response was not changed. Mediation of EDHF varies by vessel and species ²¹ but can include H₂O₂ ¹ and EETs ². However, the impaired EDHF responses in this study in mesenteric arterioles of rats infused with Ang II were independent of H₂O₂ or EET generation or action. Thus, neither H₂O₂ nor EETs caused significant vasorelaxation except at very high H₂O₂ concentrations, and the addition of catalase to metabolize H₂O₂ or the EET antagonist, 14,15-EEZE to the bath did not alter EDHF responses. The mediator of EDHF was not identified in this study.

EDHF is upregulated in blood vessels of NOS-3 knockout mice ²². We confirm that Ang II infused rats have impaired EDRF responses in mesenteric arterioles ²³. Kang et al reported that Ang II reduced the sensitivity of mesenteric vessels to Ach, as in the present study, but reduced the maximal response only after blockade of NOS. They proposed that NOS was upregulated in Ang II-infused rats to compensate for a defect in K⁺-channel mediated relaxation ⁵. However, in our study, the maximal EDRF response and the Ach-induced NO activity were reduced in vessels from Ang II infused rats. The difference may relate to the lower rate of Ang II infusion.

An endothelium dependent contraction is apparent in many models of hypertension ^4,24. EDCF responses entailed an endothelial COX-dependent production of a factor under the influence of ROS ⁶ that activates TP-Rs on VSMCs ⁴. The contractile responses of vessels from Ang II infused rats in the present study were prevented by blockade of COX-1, TP-Rs or endothelium removal but blockade of TxA₂ synthase had no significant effect. The present study did not identify the COX-1 product responsible, but prostaglandin endoperoxides have been implicated. The vascular signalling of Ang II contractile response via TP-Rs in this study may underlie the finding that TP-R knockout mice have an impaired pressor and renal vasoconstrictor response to infused Ang II ²⁵.

The kidney ²⁶ and the liver ²⁷ clear plasma ADMA and the liver clears L-arginine. Avid uptake by these organs may account for the many-fold higher arginine concentrations in the liver and ADMA concentrations in the kidney, relative to the other organs sampled. DDAH-2 is the principal isoform expressed in the rat mesenteric resistance arterioles ¹³ and DDAH-1 in the renal proximal tubules ²⁸. The down regulation by Ang II of DDAH-2 in cultured endothelial cells ²⁹ and in mesenteric resistance arterioles in this study, and of DDAH-1 in the kidney cortex in a prior study ²⁸, could therefore have reduced the metabolism of ADMA in these organs and contributed to the increased tissue levels of ADMA. Indeed, tempol prevented the angiotensin-induced reduction in DDAH-2 expression in the mesenteric vessels, which could have contributed to the prevention of an Ang II-induced increase in vascular ADMA in angiotensin-infused rats receiving tempol. DDAH activity was not assessed in these studies. However, Tain and Baylis reported that directly increasing ROS in the kidney cortex decreased DDAH activity ³⁰. Since tempol prevented the Ang II-induced increased plasma malondialdehyde and vascular ROS activity, this relates the decreased tissue DDAH-2 expression and increased tissue ADMA to oxidative stress. However, tissue ADMA also is increased by up regulation of protein arginine methyltransferases (PRMTs) and by down regulation of cationic amino acid transporters (CATs) which could have contributed to the increased tissue ADMA in this study ⁷. We reported recently that incubation of VSMCs with Ang II increased NADPH oxidase and reduced DDAH and CAT activity and increased cellular, but not medium, levels of ADMA ¹⁰. The increase in cellular ADMA was prevented by tempol or blockade of angiotensin type 1 receptors. Further studies in VSMCs transfected with p22^phox to increase NADPH oxidase directly demonstrated reduced DDAH-1 and -2 expression and activity, increased PRMT-3 expression and activity and decreased CAT-2A expression and activity and increased cellular ADMA concentrations. Thus, ROS can direct cellular metabolic pathways to increase cellular ADMA concentrations, but a reduction in CAT activity may restrain ADMA export and limit accumulation of ADMA in the extracellular fluid or plasma.

The 50% reduction in DDAH-2 expression in mesenteric vessels with Ang II in this study was similar to that produced by in vivo gene silencing ¹³ which impaired EDRF responses and NO bioactivity in mesenteric arterioles substantially. Moreover, Torondel et al ³¹ reported that overexpression of DDAH-1 or -2 in vascular endothelial cells reduced ADMA concentrations, enhanced in vitro vascular NO production and conferred resistance to administration of ADMA in DDAH heterozygote mice. The 70% increase in ADMA and 33% reduction in arginine:ADMA ratio in mesenteric vessels of angiotensin infused rats should contribute to the reduced EDRF responses, since this would reduce the substrate:inhibitor ratio for NOS. Ang II increased the mesenteric vascular arginine levels in rats given tempol, which also could have contributed to improved EDRF responses in tempol-treated rats, but the reason for this was unclear. Moreover, the increase in superoxide in the blood vessels during Ang II infusion should have enhanced NO bioinactivation ¹¹. The finding that the Ang II-induced functional defects and reduced NO and DDAH-2 expression and increased tissue levels of ADMA and ROS were all prevented by tempol, implies that an increase in ROS was an upstream event that impaired endothelial responses by coordinating these pathways for endothelial dysfunction.

Perspective

Infusions of Ang II have variable effects on plasma ADMA. Thus, prolonged Ang II infusions into mice doubled plasma ADMA in one study ⁹ whereas in others Ang II did not change plasma ADMA ^8,32, as in the present study, except at high rates of Ang II infusion that caused renal damage and reduced renal DDAH expression ⁸. We find that tissue ADMA can increase during a slow pressor Ang II infusion despite unchanged plasma levels.

Endothelial dysfunction or plasma markers of ROS ¹¹ or ADMA ³³ predict future cardiovascular events or death in high risk patients. The present study shows that tissue ADMA may not always be reflected by plasma levels, but that plasma malondialdehyde was a valid predictor of vascular ROS. A recent review concluded that the mechanisms that regulate the balances between NO and EDCF, and the processes that transform the endothelium from a protective organ to a source of vasoconstriction, proaggregatory and promitogenic responses in human hypertension are important but remain to be determined ²⁴. The present finding that tempol given to Ang II infused rats prevented the defective endothelial relaxation responses and NO bioactivity and the enhanced ROS, endothelial contractions and ADMA suggest that vascular ROS is an important component of this transformation of endothelial function. Thus, maneuvers to reduce ROS could have therapeutic potential ¹¹.