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(Hypertension. 2007;49:341.)
© 2007 American Heart Association, Inc.
Original Articles |
From the Lady Davis Institute for Medical Research (C.S., E.L.S.), Sir Mortimer B. DavisJewish General Hospital, McGill University, Montreal, Quebec, Canada; Kidney Research Center (R.M.T.), Ontario Health Research Institute, University of Ottawa, Ottawa, Ontario, Canada; Division of Cardiology (M.V.), 2nd Faculty of Medicine, University "La Sapienza," Ospedale SantAndrea and IRCCS Neuromed, Pozzilli, Italy.
Correspondence to Ernesto L. Schiffrin, Department of Medicine, Sir Mortimer B. DavisJewish General Hospital, #B-127, 3755 Cote Ste-Catherine Rd, Montreal, Quebec, Canada H3T 1E2. E-mail ernesto.schiffrin{at}mcgill.ca
| Abstract |
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Key Words: AT2 receptor angiotensin receptors human hypertension type 2 diabetes resistance arteries
| Introduction |
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Studies in cellular and animal models suggest that AT2R counteracts many AT1R actions by inducing vasodilation, antiproliferation, and apoptosis.2 We showed recently in animal models of hypertension that AT2R is upregulated and mediates vasodilation only when AT1Rs are blocked,6,7 suggesting that AT2R participates in the mechanisms whereby Ang II receptor antagonism lowers blood pressure (BP).7 Nevertheless, the role of AT2R remains unclear in human pathophysiology (eg, hypertension, diabetes, and other cardiovascular diseases), where Ang IIinduced AT1R stimulation plays a major role.
In primary hypertensive17 and high-risk hypertensive type 2 diabetic patients,18 we reported that selective AT1R antagonism improved remodeling of resistance arteries beyond BP control, which could result in improved cardiovascular outcomes. Clinical studies in diabetic patients have shown that angiotensin receptor blockers (ARBs) exert renal protective effects better than other antihypertensive drugs.1921 Thus, in humans, selective AT1R blockade may improve the structure of resistance arteries and tissue perfusion, effects that may involve an action mediated by AT2R. There is, however, no convincing evidence of AT2R functional expression in human peripheral resistance arteries in normal or pathological conditions. In this study, we investigated the hypothesis that AT2R is expressed and functionally active in peripheral resistance arteries of hypertensive type 2 diabetic patients treated with an ARB compared with similar subjects treated with a ß-blocker.
| Methods |
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Vascular Studies
The study of resistance arteries was performed by individuals unaware of the groups to which samples belonged. Small arteries (lumen diameter: 150 to 300 µm) were isolated from subcutaneous tissue immediately after the biopsy and mounted on a pressurized myograph as described previously.18 Endothelium-dependent and -independent relaxations were assessed by the dilatory responses to acetylcholine hydrochloride (1 nmol/L to 100 µmol/L) and sodium nitroprusside (10 nmol/L to 1 mmol/L), respectively, in vessels precontracted with norepinephrine (1 µmol/L). Doseresponse curves to Ang II (1 nmol/L to 1 µmol/L) in the presence of the AT1R blocker valsartan (10 µmol/L) with or without AT2R blocker PD123319 (1 µmol/L) were performed in norepinephrine-precontracted vessels.
Laser Confocal Microscopy
Resistance arteries were fixed for 30 minutes under a constant intraluminal pressure (60 mm Hg) with a solution containing 3.5% formaldehyde and 0.75% glutaraldehyde in 50 mmol/L PBS (pH 7.4).22,23 The fixative was washed from the organ bath by repeated changes with PBS (pH 7.4) and finally washed with PBS containing 0.1% Triton X-100 (pH 8.0). Arteries were incubated with 0.5% BSA/PBS for 5 minutes at 42°C. Subsequently, tissues were incubated with or without AT2R antibody (Santa Cruz Biotechnology, lot E0203) in the presence or not of excess specific AT2R blocking peptide (Santa Cruz Biotechnology, lot 1028) for 16 hours at 4°C. Tissues were washed with PBS and incubated with 200 µg/mL of Alexa Fluor 647 anti-goat IgG (Molecular Probes Inc) for 30 minutes at 37°C. For the final 30 minutes of incubation, rhodamine/phalloidin (Sigma, 10 µmol/L) was added to stain
-actin. Arteries were mounted in 1:1 glycerol/PBS (pH 7.4) on glass coverslips and studied by confocal immunofluorescence microscopy with a Zeiss LSM 510 system. The amount of AT2R present in the vessel wall was quantified by imaging (Northern Eclipse program, EMPIX Imaging Inc) and expressed as a percentage of the AT2R per total surface area.
Materials
Acetylcholine, sodium nitroprusside, and norepinephrine were obtained from Sigma Chemicals. Ang II was from Peptide International. The selective AT1R antagonist valsartan was a gift from Novartis, and PD123319 was a kind gift from Pfizer Canada. Except for valsartan, which was dissolved in 0.1 N KOH (pH 8), all of the other agents were dissolved in saline.
Data Analysis
Results are presented as mean±SEM and were analyzed by 2-way ANOVA and 1-way ANOVA followed by NewmanKeuls test, as appropriate. P<0.05 was considered statistically significant.
| Results |
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Ang II did not evoke dilation in norepinephrine-precontracted vessels from normotensive subjects and patients before the randomization (Figure 1a). Ang II did not elicit significant vasodilatory responses in arteries obtained from patients treated for 1 year with atenolol compared with those from patients before treatment (Figure 1b). In contrast, Ang II induced a dose-dependent vasodilation in precontracted resistance arteries from patients treated for 1 year with valsartan (P<0.05 versus before treatment, Figure 1c; P<0.05 versus patients treated 1 year with atenolol, Figure 1d). The AT2R antagonist PD123319 did not exert any effect on vessels from atenolol-treated patients (Figure 2a), whereas Ang IIelicited vasodilation was significantly blunted by PD123319 in the vessels from patients treated for 1 year with valsartan (Figure 2b).
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AT2Rs were expressed in resistance arteries from normotensive and hypertensive subjects at a very low density, as assessed by confocal microscopy (Figure 3a and 3b). AT2R expression increased significantly in hypertensive type 2 diabetic patient arteries only after 1-year treatment with valsartan (Figure 3a and 3b). Expression of AT2R was observed in the entire vascular wall, albeit mainly in the media (Figure 4a). Specificity of labeling of AT2R by the antibody was demonstrated by preventing antibody binding with AT2R blocking peptide (Figure 4b).
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| Discussion |
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To test the functional significance of AT2R upregulation in resistance arteries in hypertensive diabetic patients, Ang IIinduced vasodilatory responses of precontracted vessels were assessed. The isolated vessels were further exposed to valsartan ex vivo to ensuring complete AT1R blockade, allowing for examination of functional AT2R-mediated effects by Ang II. The results highlight the countervailing roles of AT1R and AT2R on Ang IIinduced vasomotor responses in humans.
Although it is clearly established that Ang II induces vasoconstriction and increased BP by stimulating AT1R,1,2 the role of AT2R on vascular responses to Ang II and on systemic BP has remained unclear in humans. It was reported recently that AT2R-mediated vasodilation occurs in the coronary microcirculation of nondiseased hearts in a cohort of subjects with a wide age range.16 As well, in healthy young subjects treated for 1 week with an AT1R antagonist, intrabrachial arterial infusion of the AT2R antagonist PD123319 resulted in increased BP, with no changes in forearm vascular responses.27
Ang II elicited dose-dependent AT2R-mediated vasodilation only in resistance arteries from hypertensive diabetic patients treated for 1 year with valsartan. This effect was associated with reduced BP in treated patients. These observations support and extend the existence of a biological "cross-talk" between Ang II receptor subtypes6,7,26,28,29 in human peripheral resistance vessels. They suggest that, in the presence of AT1R blockade, Ang II stimulates functionally active, unblocked AT2R in resistance vessels of hypertensive diabetic subjects and induces vasodilation. This effect could contribute to the mechanisms whereby ARBs lower BP.
It has been reported that Ang II induces vasodilation through AT2R-stimulated NOcGMP-dependent pathways.30,31 This occurs either directly32 or indirectly through enhanced bradykinin formation6,33 or increased endothelial NO synthase activity/expression.34 Thus, AT2R may also favorably affect endothelial function. However, in the present study, patients exhibited preserved endothelium-dependent vasodilation already at the time of randomization. This may be attributed to the fact that BP was well controlled, and
65% of the patients were being treated with an angiotensin-converting enzyme inhibitor and
50% with a calcium channels blocker,18 both of which may already have improved endothelial function.17,35
AT2R expression was increased mainly in the media of the arteries from hypertensive diabetic patients treated with valsartan. Therefore, it is also possible that Ang II can induce vasodilation by direct stimulation of AT2R on vascular smooth muscle cells. Indeed, we demonstrated recently that Ang II, through its binding with AT2R, negatively regulates the Rho/Rho kinase pathway (which is involved in vascular contraction and cell growth) in vascular smooth muscle cells and in the vasculature of hypertensive rats chronically treated with AT1R blockers.7 This effect was associated with Ang IIinduced vasodilation.
We reported recently that tight BP control with the AT1R antagonist valsartan added to other antihypertensive medications improved structural changes in resistance vessels in this cohort of hypertensive diabetic patients in whom we have now studied AT2R regulation. Equally effective BP control with the ß-blocker atenolol failed to positively influence remodeling of resistance arteries.18 Therefore, beyond BP reduction, ARBs may exert beneficial actions on vascular remodeling acting as a vasodilator by blocking AT1R. Furthermore, the functional expression of AT2R on resistance arteries of hypertensive diabetic patients treated with an ARB may also participate in these beneficial effects. In presence of AT1R antagonism, Ang II may stimulate unblocked AT2R, which could reduce vascular tone and has antigrowth36 and proapoptotic effects,37 and may function on the cell membrane as an AT1R antagonist.38 Hence, AT2R stimulation could also have participated in the effects of the ARB on vascular remodeling in that study.
Perspectives
Our study highlights the functional contribution of AT2R-mediated vasodilation to antihypertensive effects of selective AT1R blockade with ARBs in high cardiovascular risk patients. Another potentially important consequence of the role of AT2R shown in this study in hypertensive diabetic patients relates to therapeutic implications. Consistent with results of large clinical trials,1921 our findings indirectly support possible advantages of AT1R blockers compared with other antihypertensive drugs, including angiotensin-converting enzyme inhibitors (which would not provide the benefit of AT2 stimulation), in hypertensive diabetic subjects.
| Acknowledgments |
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This study was supported by grant 13570 (to E.L.S.) and a group grant to the Multidisciplinary Research Group on Hypertension, both from the Canadian Institutes of Health Research, as well as a research grant from Novartis Pharma Canada (to E.L.S.). C.S. was supported in part by a fellowship from the Italian Society of Hypertension and from Chair of Cardiology 2nd Faculty of Medicine University of Rome "La Sapienza." R.M.T. is recipient of a Canada Research Chair in Hypertension Research at the University of Ottawa, and E.L.S. is recipient of a Canada Research Chair in Hypertension and Vascular Research at McGill University.
Disclosures
R.M.T. has received honoraria <$10 000 from Bristol-Myers Squibb. E.L.S. has received honoraria <$10 000 from Bristol-Myers Squibb, Bochringer-Ingelheim, Merck, Novartis, and Speedel. The remaining authors report no conflicts.
Received August 21, 2006; first decision September 8, 2006; accepted November 19, 2006.
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