Eplerenone reduces oxidative stress and enhances eNOS in SHR: vascular functional and structural consequences.

The aim of the present study was to evaluate the effect of the aldosterone receptor antagonist eplerenone on endothelial function, oxidative stress, and structural alterations present in spontaneously hypertensive rats (SHR). To carry out the study, male SHR (18 weeks old) were treated with two doses of eplerenone (30 and 100 mg/kg/day) for 10 weeks. A group of n = 8 untreated SHR was used as a control-vehicle group, and a group of Wistar Kyoto rats (n = 8) was used as a reference of normotensive conditions. Systolic arterial pressure (SAP) was measured by the tail-cuff method. Endothelium-dependent and -independent relaxations, as well as endothelial nitric oxide synthase (eNOS) and the subunit p22phox of NAD(P)H oxidase mRNA expressions, were studied in aorta from SHR untreated or treated with eplerenone. Media/lumen ratio was also calculated in aortic preparations. In addition, levels of reduced glutathione (GSH), oxidized glutathione (GSSG), and malonyl dialdehyde (MDA) were evaluated in liver homogenates. Treatment with eplerenone reduced (p < 0.05) SAP and normalized aortic media/lumen ratio and acetylcholine relaxations. Both doses of the drug enhanced (p < 0.05) eNOS and reduced p22phox mRNA expressions. Similarly, eplerenone increased (p < 0.05) hepatic GSH/GSSG ratio, and reduced (p < 0.05) hepatic MDA levels in a comparable manner. Consequently, it could be concluded that aldosterone participates in the functional and structural vascular alterations of SHR through the diminution of nitric oxide availability and an enhancement of vascular and systemic oxidative stress.

Participation of aldosterone in the vascular inflammatory response of spontaneously hypertensive rats: role of the NFkappaB/IkappaB system.

OBJECTIVE: To investigate the participation of aldosterone in the vascular inflammatory process associated with hypertension, as well as the possible involvement of the NFkappaB/IkappaB system. METHODS: Male spontaneously hypertensive rats (SHR; 20-22 weeks old) untreated or treated with either the aldosterone receptor antagonist, eplerenone (100 mg/kg per day) or triple antihypertensive therapy (HHR: hydralazine + hydrochlorothiazide + reserpine; 20 + 7 + 0.15 mg/kg per day) were used in the study. Wistar-Kyoto rats (WKY) were used as a normotensive reference group. Aortic mRNA expression and plasma levels of interleukin (IL)-1beta, IL-6 and tumour necrosis factor alpha (TNFalpha) were measured. Likewise, the aortic expression of the nuclear factor kappaB (NFkappaB) p50 subunit precursor, p105, and its inhibitor (IkappaB) were measured. RESULTS: SHR showed higher aortic expression of IL-1beta, IL-6 and TNFalpha than WKY (P < 0.05) and higher plasma levels of IL-1beta and IL-6 than WKY (P < 0.05). Moreover, SHR also presented increased aortic expression of nuclear transcription factor NFkappaB p50 subunit precursor (p105), and a reduction of its inhibitor IkappaB. Both eplerenone and HHR decreased blood pressure to a comparable extent (P < 0.05). This effect was accompanied by a reduction in plasma levels of IL-1beta and IL-6 and aortic mRNA expression of IL-1beta, IL-6 and TNFalpha. However, the effect of eplerenone was more marked, since eplerenone-treated rats showed significantly lower inflammatory parameters than SHR receiving HHR. In addition, both antihypertensive treatments increased IkappaB mRNA expression in a similar manner, but only eplerenone reduced NFkappaB mRNA expression. CONCLUSIONS: Aldosterone, as well as an increase in haemodynamic forces produced by hypertension, participate in the vascular inflammatory process associated with hypertension in SHR. This effect seems to be mediated by enhanced vascular expression of cytokines through a modification of the NFkappaB/IkappaB system.

AT-1 receptor antagonism modifies the mediation of endothelin-1, thromboxane A2, and catecholamines in the renal constrictor response to angiotensin II.

OBJECTIVE: The present study investigated the consequences of partial AT(1) receptor blockade on the participation of catecholamines, thromboxane A(2) (TXA(2)), and endothelin-1 (ET-1) in the renal vasoconstriction induced by angiotensin II (Ang II). METHODS: For this purpose, the increase in renal perfusion pressure (RPP) produced by Ang II was studied in isolated kidneys from untreated or irbesartan-treated Wistar Kyoto and spontaneously hypertensive rats (SHR), in absence or presence of the alfa-1 receptor antagonist, prazosin, the TXA(2) receptor antagonist, ifetroban, or the ET(A)/ET(B) receptor antagonist, PD145065. RESULTS: Systolic arterial pressure (SAP) was higher (P < 0.05) in SHR than in WKY. Increases in RPP produced by Ang II were comparable in kidneys from both untreated groups. Treatment with irbesartan reduced SAP and RPP in both strains in a comparable extent. Presence of prazosin, ifetroban, or PD145065 in perfusion media reduced (P < 0.05) Ang II maximal response in all groups. Maximal inhibition of Ang II-induced vasoconstriction produced by the 3 antagonists was comparable in untreated WKY, but that of ifetroban and PD145065 was lower (P < 0.05) than that of prazosin in untreated SHR. Maximal inhibition of Ang II-induced vasoconstriction produced by the 3 antagonists was comparable in WKY treated with irbesartan, and not different to that observed in untreated WKY. Maximal inhibitory effect of the 3 antagonists was higher (P < 0.05) in treated than in untreated SHR. CONCLUSION: The study further supports the importance of catecholamines, TXA(2), and ET-1 as mediators of the renal vasoconstriction induced by Ang II in both normotensive and hypertensive rats. Hypertensive conditions appeared to reduce the participation of TXA(2) and ET-1 in Ang II-induced vasoconstriction when compared with normotensive conditions. Chronic partial blockade of AT(1) receptors did not affect the participation of catecholamines, TXA(2), and ET-1 in normotensive rats, but increased the participation of the 3 mediators in SHR. This suggests that when AT(1) receptors are partially blocked, other vasoconstrictor factors could exert part of the renal vasoconstrictor effects of Ang II.

Valsartan improves fibrinolytic balance in atherosclerotic rabbits.

OBJECTIVES: To examine the long-term effects of the angiotensin type I (AT1) receptor antagonist, valsartan, on fibrinolytic balance, coagulation parameters, endothelial function and structural alterations in atherosclerotic rabbits. METHODS: Animals were submitted to a 1% cholesterol-enriched diet for 10 weeks. Half of the animals were treated with valsartan (3 or 10 mg/kg per day). Systolic arterial pressure was directly measured in awake rabbits. Tissue plasminogen activator (t-PA) and tissue plasminogen activator inhibitor (PAI-1) activities were measured. Plasma concentrations of cholesterol, D-dimer, factor VIII and fibrinogen, as well as thrombin time, were also determined. Responses to acetylcholine, sodium nitroprusside and angiotensin II were evaluated in aortic rings. Morphometric analysis of aortic segments was also performed to calculate atherosclerotic lesion. RESULTS: Cholesterol-fed rabbits presented systolic arterial pressure levels comparable to controls. These animals presented aortic atherosclerotic lesions. Treatment with valsartan did not alter plasma cholesterol levels or arterial pressure in any group. Acetylcholine-induced relaxations and D-dimer and t-PA activity were lower (P < 0.05) in atherosclerotic than in normal rabbits. In contrast, PAI-1 activity was higher (P < 0.05) in atherosclerotic rabbits than in controls. Valsartan increased (P < 0.05) acetylcholine-induced relaxations, D-dimer concentration and t-PA activity, and reduced intimal thickening and PAI-1 activity in cholesterol-fed rabbits. Fibrinogen concentrations and factor VIII concentrations were lower (P < 0.05) and thrombin time was higher (P < 0.05) in atherosclerotic rabbits compared to controls. Valsartan did not affect factor VIII in any group, but reduced fibrinogen levels only in hypercholesterolemic rabbits. Valsartan 10 mg/kg per day reduced (P < 0.05) thrombin time in cholesterol-fed rabbits. CONCLUSIONS: Impairment of fibrinolytic balance, associated with atherosclerosis in rabbits, appears to be related with angiotensin II via AT1receptors. The beneficial effect of valsartan on fibrinolysis seems to be related to the concomitant amelioration of endothelial dysfunction and reduction of intimal thickening, further supporting the importance of the blockade of angiotensin II actions to prevent thrombotic alterations associated with atherosclerosis.

Role of endothelin-1 and thromboxane A2 in renal vasoconstriction induced by angiotensin II in diabetes and hypertension.

BACKGROUND: Interactions among angiotensin II (Ang II), endothelin-1 (ET-1) and thromboxane A2 (TXA2) may play an important role in the regulation of renal function. The present study investigated the participation of TXA2 and ET-1 in the increase in renal vascular resistances (RVR) induced by Ang II, as well as the consequences of diabetes, hypertension, and the combination of both on this response. METHODS: Isolated kidneys from male normoglycemic or streptozotocin-induced diabetic Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were used. The increase in perfusion pressure (PP) produced by Ang II was studied in the absence or presence of the TXA2 receptor antagonist, ifetroban, or the ETA/ETB receptor antagonist, PD145065. RESULTS: Systolic arterial pressure (SAP) was higher in SHR than in WKY, but diabetic rats (D) from each strain showed lower SAP values than their respective non-diabetic rats. Basal renal PP was higher in WKY and SHR than in WKY-D and SHR-D. Increases in renal PP produced by Ang II were comparable in the kidneys from all groups. Either ifetroban or PD145065 reduced the maximal Ang II response in all animals. The maximal inhibitory effect of ifetroban was higher (P<0.05) in WKY than in the other groups. However, the maximal inhibitory effect of PD145065 was lower in SHR than in the other groups. CONCLUSION: This study supports a role for ET-1 and TXA2 as mediators of the increase in renal vascular resistance produced by Ang II. These results indicate that the participation of ET-1 in the renal vasoconstriction produced by Ang II was reduced under hypertensive conditions, and that of TXA2 was reduced by both diabetes and hypertension.

Effect of AT1 receptor antagonism on vascular and circulating inflammatory mediators in SHR: role of NF-kappaB/IkappaB system.

We investigated the role of angiotensin II in vascular and circulating inflammatory markers in spontaneously hypertensive rats (SHR). IL-1beta, IL-6, and TNF-alpha aortic mRNA expression and plasma levels were measured in adult SHR untreated or treated with the angiotensin II receptor antagonist candesartan (2 mg.kg(-1).day(-1)) or antihypertensive triple therapy (TT; in mg.kg(-1).day(-1): 20 hydralazine + 7 type 1 hydrochlorothiazide + 0.15 reserpine) for 10 wk. Likewise, aortic expression of NF-kappaB p50 subunit precursor p105 and its inhibitor (IkappaB) were measured. Age-matched Wistar-Kyoto rats (WKY) served as normotensive reference. High blood pressure levels were associated with increased (P < 0.05) aortic mRNA expression of IL-1beta, IL-6, and TNF-alpha. Hypertension was also accompanied by increased IL-1beta and IL-6 plasma levels. No differences were observed in circulating TNF-alpha levels between SHR and WKY. SHR presented elevated aortic mRNA expression of the transcription factor NF-kappaB and reduction in its inhibitor, IkappaB. Candesartan decreased (P < 0.05) blood pressure levels, aortic mRNA expression of IL-1beta, IL-6, and TNF-alpha, and (P < 0.05) IL-1beta and IL-6 plasma concentration. However, although arterial pressure decrease was comparable for the treatments, TT only partially reduced the increments in inflammatory markers. In fact, candesartan-treated rats showed significantly lower levels of circulating and vascular inflammatory markers than TT-treated animals. The treatments increased IkappaB mRNA expression similarly. However, only candesartan reduced NF-kappaB mRNA expression. In summary, 1) SHR presented a vascular inflammatory process; 2) angiotensin II, and increased hemodynamic forces associated with hypertension, seems to be involved in stimulation of inflammatory mediators through NF-kappaB system activation; and 3) reduction of inflammatory mediators produced by candesartan in SHR could be partially due to both downregulation of NF-kappaB and upregulation of IkappaB.

Comparison between the effects of mixed dyslipidaemia and hypercholesterolaemia on endothelial function, atherosclerotic lesions and fibrinolysis in rabbits.

We compared the impact of hypercholesterolaemia and mixed dyslipidaemia on vascular function, vascular structure and fibrinolytic balance in rabbits. To this end, vascular reactivity was studied in aortic rings from rabbits fed a control diet, a diet containing 0.5% cholesterol+14% coconut oil (mixed dyslipidaemia) or a diet containing 1% cholesterol (hypercholesterolaemia) for 12-14 weeks. Morphometric analysis of aorta was also performed and plasminogen activator inhibitor-1 (PAI-1) as well as tissue-type plasminogen activator (t-PA) plasma activities were measured. Both diets induced a similar increase in cholesterol plasma levels, although triacylglycerols (triglycerides) were increased in animals with mixed dyslipidaemia. Hypercholesterolaemia was associated with intimal thickening, reduction in acetylcholine-induced relaxation ( P <0.05) and increased vasoconstriction induced by acetylcholine+ N (G)-nitro-L-arginine methyl ester (L-NAME) when compared with controls ( P <0.05). These effects were more marked ( P <0.05) in animals with mixed dyslipidaemia. Incubation with ifetroban, a thromboxane A(2)/prostaglandin H(2) receptor antagonist, increased acetylcholine-induced relaxation ( P <0.05) and reduced acetylcholine+L-NAME contraction ( P <0.05) in both diet groups. In contrast, the presence of PD 145, an endothelin (ET)(A)/ET(B) receptor antagonist, exerted these effects only in rabbits with mixed dyslipidaemia. Both hypercholesterolaemia and mixed dyslipidaemia induced a similar increase in PAI-1 and a similar decrease in t-PA plasma activities. These data suggest that hypertriglyceridaemia can increase the deleterious effects of hypercholesterolaemia on endothelial function and vascular structure. This additional harmful effect exerted by triacylglycerols on endothelial function could, in part, be mediated by ET.

Effect of AT1 receptor blockade on hepatic redox status in SHR: possible relevance for endothelial function?

The study investigated whether the amelioration of endothelial dysfunction by candesartan (2 mg.kg-1.day-1; 10 wk) in spontaneously hypertensive rats (SHR) was associated with modification of hepatic redox system. Systolic arterial pressure (SAP) was higher (P < 0.05) in SHR than in Wistar-Kyoto rats (WKY) and was reduced (P < 0.05) by candesartan in both strains. Acetylcholine (ACh) relaxations were smaller (P < 0.05) and contractions induced by ACh + NG-nitro-l-arginine methyl ester (l-NAME) were greater (P < 0.05) in SHR than in WKY. Treatment with candesartan enhanced (P < 0.05) ACh relaxations in SHR and reduced (P < 0.05) ACh + l-NAME contractions in both strains. Expression of aortic endothelial nitric oxide synthase (eNOS) mRNA was similar in WKY and SHR, and candesartan increased (P < 0.05) it in both strains. Aortic mRNA expression of the subunit p22phox of NAD(P)H oxidase was higher (P < 0.05) in SHR than in WKY. Treatment with candesartan reduced (P < 0.05) p22phox expression only in SHR. Malonyl dialdehyde (MDA) levels were higher (P < 0.05), and the ratio reduced/oxidized glutathione (GSH/GSSG) as well as glutathione peroxidase activity (GPx) were lower (P < 0.05) in liver homogenates from SHR than from WKY. Candesartan reduced (P < 0.05) MDA and increased (P < 0.05) GSH/GSSG ratio without affecting GPx. Vessel, lumen, and media areas were bigger (P < 0.05) in SHR than in WKY. Candesartan treatment reduced (P < 0.05) media area in SHR without affecting vessel or lumen area. The results suggest that hypertension is not only associated with elevation of vascular superoxide anions but with alterations of the hepatic redox system, where ANG II is clearly involved. The results further support the key role of ANG II via AT1 receptors for the functional and structural vascular alterations produced by hypertension.

Synergistic effect of angiotensin-converting enzyme (ACE) and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibition on inflammatory markers in atherosclerotic rabbits.

In the present study, we compared the effect of atorvastatin (1 mg.kg(-1).day(-1)) and quinapril (0.5 mg.kg(-1).day(-1)) alone or in combination on inflammatory markers, endothelial function, intimal thickening and fibrinolytic balance in rabbits fed with either a control diet or a diet containing 1% (v/v) cholesterol for 12 weeks. Atorvastatin alone or in combination partially prevented the increase in cholesterol plasma levels observed in rabbits fed with the cholesterol-rich diet, but did not modify blood pressure levels. Quinapril administration did not alter any of these parameters in any group. Hypercholesterolaemia increased plasma levels of interleukin-1beta, interleukin-6, interferon-gamma and C-reactive protein, reduced acetylcholine-induced relaxation and produced intimal thickening. Likewise, atherosclerotic rabbits had reduced plasma tissue-type plasminogen activator activity and D-dimer levels and an increase in plasminogen-activator inhibitor-1 activity. Both drugs enhanced acetylcholine-induced relaxation, reduced intimal thickening and improved fibrinolytic balance in atherosclerotic rabbits in a similar manner. Their combination did not induce additive effects on these parameters. However, only the combination of both drugs was able to prevent the increase in inflammatory markers induced by hypercholesterolaemia. In summary, these data suggest that quinapril and atorvastatin had comparable beneficial effects on the alterations of vascular function and structure as well as fibrinolytic balance in atherosclerotic rabbits. In addition, the combination of atorvastatin and quinapril exerts a synergistic effect on inflammatory markers, which individual treatment, at the doses used, was not able to modify.