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Abstract

Introduction:

The angiotensin II (Ang II) type 1 receptor exerts pro-atherogenic action by augmenting oxidative stress, whereas the Ang II type 2 receptor (AT₂)-mediated effect on atherosclerosis remains controversial.

Materials and methods:

AT₂ transgenic (AT₂-Tg) mice, which overexpress AT₂ in their vascular smooth muscle cells, were crossed with apoE-deficient (apoE^-/-) mice to generate AT₂ transgenic apoE^-/- mice (AT₂-Tg/apoE^-/-).

Results:

A subpressor dose of Ang II infusion exaggerated atherosclerosis development in apoE^-/- mice, which was markedly suppressed in AT₂-Tg/apoE^-/- mice. Inhibitors of nitric oxide (NO) synthase (L-NAME) or bradykinin type 2 receptor completely abolished AT₂-mediated anti-atherogenic actions. The vascular cell adhesion molecule-1 expression levels and degree of monocyte/macrophage accumulation in the intima were also considerably reduced in AT₂-Tg/apoE^-/- mice; these phenomena were completely reversed by L-NAME treatment. Ang II infusion significantly enhanced the accumulation of dihydroethidium-positive mononuclear cells in the intima and mRNA expression levels of Nox2, a phagocytic cell-type NADPH oxidase subunit in apoE^-/- mice, which was completely inhibited in AT₂-Tg/apoE^-/- mice.

Conclusions:

Vascular AT₂ stimulation exerts anti-atherogenic actions in an endothelial kinin/NO-dependent manner, and its anti-oxidative effect is likely to be exerted by inhibiting the accumulation of superoxide-producing mononuclear leukocytes.

Keywords

Angiotensin receptor atherosclerosis nitric oxide oxidative stress

Introduction

Angiotensin II (Ang II) has been shown to promote atherosclerosis^1,2 by activating various signaling pathways and augmenting oxidative stress via type 1 receptor (AT₁) activation,^3,4 which leads to increased expressions of the redox-sensitive genes involved in atherogenesis.^5,6 In contrast, the effect of Ang II type 2 receptor (AT₂) on atherogenesis remains controversial. Daugherty et al. demonstrated that AT₂ antagonist treatment exaggerated Ang II-induced atherosclerosis in apoE^-/- mice⁷; however, studies using AT₂-deficient mice showed conflicting results. Daugherty et al. showed that AT₂ deficiency did not affect atherosclerosis in low density lipoprotein (LDL) receptor-deficient mice,⁸ whereas Iwai et al. reported that the atherosclerotic lesion area was significantly enhanced in AT₂/apoE double-deficient mice through the exacerbation of oxidative stress.⁹ These results raise concerns about the AT₂-medited anti-atherogenic effect of long-term treatment with Ang II receptor blockers (ARB) or compound 21 (C21), a recently developed orally active non-peptide and selective AT₂ agonist.¹⁰

The expression of AT₂ is known to be restricted in the adult cardiovascular system, with lower expression levels relative to the AT₁ receptor, while it is up-regulated in various pathological conditions including post-myocardial infarction, heart failure, and arterial injury.^11–13 It was recently shown that the AT₂ is expressed in the muscular medial layer of the aorta and in endothelial cells of resistant arteries,¹⁴ and it is also up-regulated in the aortas of apoE^-/- mice on an atherogenic diet.^9,15 We previously generated AT₂ transgenic (AT₂-Tg) mice featuring targeted AT₂ overexpression in the vascular smooth muscle cells (VSMCs) and demonstrated that AT₂ in VSMCs promotes intracellular acidosis by blocking the amiloride-sensitive Na⁺/H⁺ exchanger, leading to kininogenase activation followed by bradykinin release from VSMCs, resulting in endothelial nitric oxide (NO)-dependent vasodilation and an anti-pressor response to Ang II.¹⁶ These findings lead to the hypothesis that AT₂ in the aortic medial layer exerts its anti-atherogenic action through endothelial bradykinin/NO system activation.

In this study, we crossed AT₂-Tg mice with apoE^-/- mice to generate vascular AT₂-overexpressing apoE^-/- (AT₂-Tg/apoE^-/-) mice in an effort to elucidate the involvement of the kinin/NO system in the observed AT₂-mediated anti-atherogenic action. We found that vascular AT₂ activation exerts its anti-atherogenic action in an endothelial kinin/NO-dependent manner accompanied by decreased oxidative stress through decreased accumulation of superoxide-producing mononuclear leukocytes. Our findings support the notion that direct AT₂ activation could be a potential therapeutic option for preventing the development of atherosclerotic cardiovascular diseases.

Materials and methods

Animal preparation

AT₂-Tg mice (C57BL/6) overexpressing the AT₂ under control of the α-smooth muscle actin promoter were generated as described previously.¹⁶ Homozygous AT₂-Tg mice were crossed with homozygous apoE^-/- mice (C57BL/6) to yield double heterozygous mice. These heterozygous littermates were bred with apoE^-/- mice to establish AT₂-Tg/apoE^-/- mice (backcrossed>10 times). Male apoE^-/- mice and AT₂-Tg/apoE^-/- mice were weaned at 5 weeks of age onto a high-cholesterol diet (21% fat, 0.125% cholesterol; Oriental Yeast Co., Tokyo, Japan) that was maintained for 8 weeks. Starting at the age of 8 weeks, a subpressor dose of Ang II (100 ng/kg/min) or phosphate-buffered saline (PBS) was administered for 4 weeks using an osmotic minipump. Before the implantation of osmotic minipumps, mice were anesthetized with a single intraperitoneal injection of ketamine (50 mg/kg) and xylazine (8 mg/kg). Sufficient anesthesia depth was confirmed by the lack of a tail pinch response. Additional groups of 8-week-old apoE^-/- and AT₂-Tg/apoE^-/- mice were treated with either L-NG-nitroarginine methyl ester (L-NAME; 1 mg/ml in drinking water) or the bradykinin receptor inhibitor icatibant (70 mg/kg/day, osmotic minipump) in parallel with the Ang II infusion.

Before the aortic tissues were isolated, the mice were sacrificed by transcardiac perfusion under terminal anesthesia by intraperitoneal injection of pentobarbital (200 mg/kg). All investigations conformed to the Guidelines for Animal Experiments of the Kyoto Prefectural University of Medicine. The experiments were also approved by a local university ethics review board.

Membrane preparation and receptor assay

Membrane fractions were prepared from pooled aortic samples and incubated with different concentrations (0.05–1 nM) of [¹²⁵I]Sar1, Ile8-Ang II, or [¹²⁵I]CGP42112A for 120 min at 20°C for the saturation experiment as described previously.¹⁷ Specific binding of [¹²⁵I]Sar1, Ile8-Ang II, and [¹²⁵I]CGP42112A was determined from the difference between counts in the absence of 10 µmol/l AT₁ antagonist (losartan) and the presence of 1 µmol/l AT₂ antagonist (CGP42112A). K_d and B_max values were estimated by Rosenthal analysis of the saturation data, while the AT₁ and AT₂ densities were calculated from the B_max values.

Hemodynamic analysis

Mean blood pressure and heart rate were measured under conscious and unrestrained conditions using a programmable sphygmomanometer (BP-98A; Softron, Tokyo, Japan).

Plasma lipid analysis

Total cholesterol and triglyceride concentrations were determined using an automated chemistry analyzer (Wako Chemicals Co., Tokyo, Japan). LDL-cholesterol levels were quantified by an enzymatic reaction using a commercially available kit (Daiichi Pure Chemicals Co., Tokyo, Japan).

Quantitative measurement of atherosclerotic lesions

The mice were euthanized at 12 weeks of age and the atherosclerotic lesions were analyzed as described previously.¹⁸ Image analysis was performed on Oil Red O-stained aortas using Scion Imaging software. The aortic lesion area in each animal was measured as the percentage of lesion area per total aortic area.

Immunohistochemistry

For immunohistochemical analysis and superoxide product evaluation, Ang II was administered for 2 weeks to the apoE^-/- and AT₂-Tg/apoE^-/- mice from the age of 8 weeks. The aortic arch was rapidly removed after PBS perfusion, embedded in optimal cutting temperature compound, and quick-frozen in liquid nitrogen. The atherosclerotic lesions in the aortic arch region were examined at five locations at 100 µm intervals, and nine serial 6 µm-thick sections were prepared from each location, stained immunohistochemically with fluorescein isothiocyanate (FITC)- or tetramethyl rhodamine isothiocyanate (TRITC)-conjugated antibodies against vascular cell adhesion molecule-1 (VCAM-1; clone 429, Pharmingen, San Diego, CA) and monocyte/macrophage antibody-2 (MOMA-2; Serotec, Oxford, UK), and then observed using a confocal microscope (FLUOVIEW FV300; Olympus, Tokyo, Japan). As a negative control, non-immune immunoglobulin and FITC- or TRITC-conjugated secondary antibodies were used. Positive VCAM-1 and MOMA-2 staining was evaluated using image-analysis software. The percentage coverage of VCAM-1 in the total aortic area and the MOMA-2 staining area were assessed in five sections from six animals in each group.

In situ detection of superoxide anions

Fresh-frozen cross-sections of the ascending aorta (30 µm each) were incubated with dihydroethidium (DHE) (1 µM) in PBS for 30 min at 37°C in a humidified chamber protected from light.¹⁹ To detect the ethidium, samples were examined using a fluorescence microscope equipped with a computer-based imaging system. The fluorescence intensity was pixilated and quantified using Scion Image software.

Real-time polymerase chain reaction

The thoracic aortas of the mice were dissected and the total RNA was isolated. Real-time polymerase chain reaction (RT-PCR) was performed using a LightCycler ST300 (Roche Applied Science, Indianapolis, IN) with SYBR Green PCR Master Mix and RT-PCR (Applied Biosystems, Foster City, CA) as described previously.²⁰ The dissociation curves were monitored to check for the aberrant primer dimers formation. PCR-amplified products were electrophoresed on 2% agarose gels to confirm the presence of a single band. Data are expressed as levels relative to apoE^-/- mice with PBS treatment.

Nicotinamide adenine dinucleotide phosphate oxidase activity

Protein samples were extracted from aortas after 1 week of Ang II infusion, and incubated for 30 min in Krebs-HEPES buffer at 37°C. The rings were transferred to scintillation vials containing 100 µmol/l L-012 in Krebs-HEPES buffer and incubated for 5 min at 37°C in the dark. After incubation, chemiluminescence was measured using a luminometer (Lumat LB 9507; Berthold Technologies Ltd) over a period of 10 min at 1 min intervals. After measurement, 100 µmol/l nicotinamide adenine dinucleotide phosphate (NADPH) (Sigma Chemical Co, St Louis, MO) was added and the rings were further incubated at 37°C for 60 min. L-012 chemiluminescence was expressed as relative light units per milligram of dry tissue weight per minute. NADPH oxidase activity was quantified from the absorbance with or without NADPH.

Measurement of Ca²⁺-dependent NO synthase activity

Protein samples were extracted from the aortas after 1 week of Ang II infusion. NO synthase (NOS) activity was measured using the conversion rate of radio-labeled L-[³H] citrulline from L-[³H] arginine (Amersham, Little Chalfont, UK).²¹ Duplicate incubations were performed for 60 min at room temperature for each sample in the presence or absence of ethylene glycol tetraacetic acid (2 mmol/l) to determine the Ca²⁺-dependent NOS activity.

Statistical analysis

All data are expressed as means ± SE. Mean values were compared using analysis of variance. If a statistically significant effect was found, Scheffe’s F test was performed to detect the difference between groups. Values of p<0.05 were considered statistically significant.

Results

Expression of AT₂ in the aortas of AT2-Tg/apoE^-/- mice

We generated five different AT₂-Tg lines and then crossed AT₂-Tg901 and AT₂-Tg902 mice, which expressed aortic AT₂ most abundantly, with apoE^-/- mice. As the findings obtained from AT₂-Tg901/apoE^-/- and AT₂-Tg902/apoE^-/- mice were similar, we here described the result from AT₂-Tg901/apoE^-/- that expressed aortic AT₂ (10.1 ± 1.8 fmol/mg protein) (Supplemental Figure 1) more abundantly than AT₂-Tg902/apoE^-/- mice (8.9 ± 1.2 fmol/mg protein). The proportion of aortic AT₂ relative to AT₁ was 33% ± 0.1%, identical to that in AT₂-Tg901 mice aortas (39% ± 0.2 %). AT₂ was not detected in the aortas of apoE^-/- mice, and there were no significant differences in aortic AT₁ receptor density between apoE^-/- and AT₂-Tg901/apoE^-/- mice (Supplemental Figure 1). The K_d value (nmol/l) of AT₂ in the aorta from AT₂-Tg901/apoE^-/- mice was 0.39 ± 0.05, similar to that in AT₂-Tg901 (0.31 ± 0.01). These animals had similar weights relative to their control littermates (designated as apoE^-/- mice), and there was no evidence of obvious morphological changes of the aorta.

Vascular AT₂ activation inhibits atherosclerosis development in apoE^-/- mice

We first compared the atherosclerotic lesion area between apoE^-/- and AT₂-Tg/apoE^-/- mice fed a high-cholesterol diet for 8 weeks; however, no significant difference was observed between groups. We next examined the effect of a subpressor dose of Ang II (100 ng/kg/min) on atherosclerotic lesion development. Atherosclerotic lesion area was markedly increased in apoE^-/- mice (from 3.0% ± 0.4% to 14.5% ± 1.6%, p<0.001); however, Ang II-induced atherosclerotic lesion development was substantially reduced by 53% in AT₂-Tg/apoE^-/- mice (p<0.05) (Figures 1(a) and 1(b)), suggesting that activation of the vascular AT₂ exerts anti-atherogenic actions. Hemodynamic parameters and lipid profiles did not differ between the two groups (Supplemental Figures 2A and 2B).

Figure 1.

Vascular angiotensin II (Ang II) type 2 receptor (AT2) inhibits Ang II-induced atherosclerotic lesion formation. (A) Representative photographs of en face Oil Red O-stained aortas in apoE-deficient (apoE-/-) and AT2 transgenic (AT2-Tg)/apoE-/- (AT2-Tg/apoE-/-) mice treated with phosphate-buffered saline (PBS) or Ang II (100 ng/kg/min). The scale bar shows 3 mm intervals. (B) Quantitative analysis showing a significant reduction in the percentage of lesion coverage in the aorta in Ang II-treated AT2-Tg/apoE-/- mice. Values are the mean ± SE for at least 8 mice in each group. *p<0.01 vs. PBS-treated apoE-/- mice. #p<0.05 vs. PBS-treated AT2-Tg/apoE-/- mice. †p<0.05 vs. Ang II-treated apoE-/- mice.

Blockade of the bradykinin/NO system by L-NAME or icatibant abolishes AT₂-mediated anti-atherogenic actions

We previously demonstrated that targeted overexpression of the AT₂ in VSMCs exerted an antipressor response to Ang II through a kinin/NO-dependent mechanism.¹⁶ To elucidate the involvement of the kinin/NO system in AT₂-mediated anti-atherogenic actions, L-NAME or icatibant was administered in combination with the Ang II infusion. Co-treatment with L-NAME did not affect the Ang II-induced atherosclerotic lesions in apoE^-/- mice; however, the lesion area in L-NAME-treated AT₂-Tg/apoE^-/- mice was significantly increased to a similar extent as that seen in apoE^-/- mice (13.6% ± 1.0% vs. 12.1% ± 1.6%, apoE^-/- vs. AT₂-Tg/apoE^-/-; p=n.s.) (Figures 2(a) and 2(b)). Similarly, the AT₂-mediated anti-atherogenic action observed in AT₂-Tg/apoE^-/- mice was completely reversed by icatibant treatment. Considering that the bradykinin type 2 receptor is exclusively expressed in the endothelium, these results suggest that the endothelial bradykinin/NO system is likely to be involved in AT₂-mediated anti-atherogenic action.

Figure 2.

L-NG-nitroarginine methyl ester (L-NAME) or icatibant abolishes vascular angiotensin II type 2 receptor (AT2)-mediated anti-atherogenic effects. (a) Representative photographs of en face Oil Red O-stained aortas in apoE-/- and AT2 transgenic/apoE-/- (AT2-Tg/ apoE-/-) mice treated with L-NAME or icatibant in parallel with Ang II infusion. L-NAME (1 mg/ml) was added in the drinking water of mice in parallel with Ang II (100 ng/kg/min). Icatibant (70 µg/kg per day) was co-infused with Ang II using an osmotic pump. The scale bar shows 3 mm intervals. (b) Quantitative analysis showing a significant reduction of Ang II-induced atherosclerotic lesions in AT2-Tg/apoE-/- mice, a phenomenon that was completely reversed by L-NAME or icatibant treatment. Values are the mean ± SE for at least 8 mice in each group. *p<0.05 vs. phosphate-buffered saline (PBS)-treated apoE-/-. #p<0.05 vs. PBS-treated AT2-Tg/apoE-/- mice. †p<0.05 vs. Ang II-treated apoE-/- mice. ¶p<0.05 vs. Ang II-treated AT2-Tg/apoE-/- mice.

Vascular AT₂ activation increases Ca²⁺-dependent NOS activity in apoE^-/- mice

To further elucidate the activation of an endothelial bradykinin/NO system in AT₂-Tg/apoE^-/- mice, Ca²⁺-dependent NOS activity in the aorta was examined after 1 week of Ang II infusion, in which few adhesive mononuclear cells were observed in the intima (data not shown). In the absence of Ang II infusion, Ca²⁺-dependent NOS activity did not differ between the two groups of mice (Figure 3). In contrast, Ang II infusion into AT₂-Tg/apoE^-/- mice markedly increased Ca²⁺-dependent NOS activity (67% increase vs. PBS infusion, p<0.01), but had no effect on that in apoE^-/- mice. This finding further supports the notion that the AT₂-mediated enhancement of NOS activation and the subsequent NO production through an endothelial kinin/NO-dependent mechanism play a critical role in AT₂-mediated anti-atherogenic action.

Figure 3.

Vascular angiotensin II type 2 receptor (AT2) activation increases Ca2+-dependent nitric oxide synthase (NOS) activity in apoE-/- mice. One week of Ang II infusion significantly enhanced Ca2+-dependent NOS activity in the aortas of angiotensin II type 2 receptor (AT2) transgenic/apoE-/- (AT2-Tg/apoE-/-) mice. The bar graphs represent the mean ± SE of the conversion rate from [3H]-arginine to [3H]-citrulline (pmol/min/mg protein). At least 4 mice were tested in each group. *p<0.05 vs. vessels from phosphate-buffered saline (PBS)-treated AT2-Tg/apoE-/- mice. #p<0.01 vs. vessels from Ang II-treated apoE-/- mice.

Vascular AT₂ inhibits Ang II-induced VCAM-1 expression and monocyte/macrophage accumulation

In the early stage of atherosclerosis, activated endothelial cells expressing various kinds of adhesion molecules such as VCAM-1 play a critical role in the accumulation of mononuclear cells within the intima.²² Since endothelium-derived NO has been shown to inhibit adhesion molecule expression,²³ we examined VCAM-1 expression levels and monocyte/macrophage accumulation by immunochemistry after 2 weeks of Ang II infusion. In the absence of Ang II, the VCAM-1 expression was equivalent between the two groups of mice. Consistent with the previous data,²⁴ Ang II infusion significantly increased the expression of VCAM-1 on the inner surface and medial wall in apoE^-/- mice, whereas it was markedly inhibited in AT₂-Tg/apoE^-/- mice (Figures 4(a) and 4(b)). Similarly, Ang II-induced monocyte/macrophage accumulation in the luminal surface was significantly decreased in AT₂-Tg/apoE^-/- mice compared with apoE^-/- mice (Figures 4(c) and 4(d)), suggesting that augmented NO production inhibits VCAM-1 expression, resulting in attenuated accumulation of monocytes/macrophages in AT₂-Tg/apoE^-/- mice.

Figure 4.

Vascular angiotensin II type 2 receptor (AT2) inhibits Ang II-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and monocyte/macrophage accumulations. (A) Representative positive VCAM-1 stained aortic arch sections from mice treated with 2 weeks of Ang II infusion (magnification ×200; insets, magnification ×400). L: lumen, M: media. (B) Quantitative analysis showing a significant increase in Ang II-induced expression of VCAM-1 in both the intimal and medial layers of apoE-/- mice. The same was markedly suppressed in AT2 transgenic/apoE-/- (AT2-Tg/apoE-/-) mice. Values are the mean ± SE for at least 6 mice in each group. *p<0.01 vs. vessels from phosphate-buffered saline (PBS)-treated apoE-/- mice. #p<0.01 vs. vessels from Ang II-treated apoE-/- mice. (C) Representative monocyte/macrophage antibody-2 (MOMA-2) positive-stained aortic arch sections from mice treated with 2 weeks of Ang II infusion (magnification ×100, and insets, magnification ×300). L: lumen, M: media. (D) Quantitative analysis showing a significant increase in Ang II-induced accumulation of MOMA-2 positive cells attached to the intimal surfaces in apoE-/- mice but suppression in AT2-Tg/apoE-/- mice. Values are the mean ± SE for at least 6 mice in each group. *p<0.05 vs. vessels from PBS-treated apoE-/- mice. #p<0.01 vs. vessels from Ang II-treated apoE-/- mice.

We also examined the effects of L-NAME on Ang II-induced VCAM-1 expression and monocyte/macrophage accumulation. L-NAME treatment further increased Ang II-induced VCAM-1 expression in both apoE^-/- and AT₂-Tg/apoE^-/- mice; however, the extent was much larger in AT₂-Tg/apoE^-/- mice than in apoE^-/- mice. Consequently, there was no significant difference in VCAM-1-positive area between apoE^-/- and AT₂-Tg/apoE^-/- mice after L-NAME treatment (Supplemental Figure 3A). Similarly, L-NAME treatment markedly augmented Ang II-induced monocyte/macrophage accumulation in AT₂-Tg/apoE^-/- mice to the same extent as that in apoE^-/- mice (Supplemental Figures 3B and 3C). These findings further supported the current hypothesis that vascular AT₂ overexpression exerts anti-atherogenic action through an endothelial kinin/NO system.

Vascular AT₂ attenuates Ang II-induced oxidative stress by inhibiting the accumulation of superoxide-producing mononuclear leukocytes

To elucidate the manner in which AT₂ counteracts AT₁-mediated oxidative stress, cell-specific generation of superoxide anions was examined. In the absence of Ang II, the fluorescence intensity of DHE-positive mononuclear cells in the intima did not differ between apoE^-/- and AT₂-Tg/apoE^-/- mice (Figures 5(a) and 5(b)). In contrast, 2 weeks of Ang II infusion into apoE^-/- mice significantly increased the fluorescence intensity, particularly in the intima, which was completely inhibited in AT₂-Tg/apoE^-/- mice. Consistent with this finding, the mRNA expression level of Nox2, phagocytic NADPH oxidase,²⁵ was markedly increased in apoE^-/- mice (5.1-fold vs. PBS; p<0.05) after Ang II infusion (Supplemental Figure 4A), but was significantly reduced in AT₂-Tg/apoE^-/- mice, suggesting that AT₂-mediated anti-oxidative action is mostly exerted by inhibiting the accumulation of superoxide-producing mononuclear leukocytes.

Figure 5.

Vascular angiotensin II type 2 receptor (AT₂) attenuates Ang II-induced oxidative stress by inhibiting the accumulation of superoxide-producing mononuclear leukocytes. (A) Representative photographs showing dihydroethidium (DHE)-positive mononuclear cells attached to the intimal layer (magnification ×600). Arrows indicate DHE-positive mononuclear cells attached to the intimal layer. M: media, L: lumen. (B) Quantitative analysis showing Ang II-induced augmentation of superoxide production in apoE-deficient (apoE^-/-) mice, a phenomenon that was profoundly attenuated in AT₂ transgenic/apoE^-/- (AT₂-Tg/apoE^-/-) mice. Superoxide levels were calculated as the ratio (fold increase) to the control values obtained from sequential unstained sections from each mouse. The bar graphs represent the mean ± SE relative to phosphate-buffered saline (PBS)-treated apoE^-/- mice set at 100%. At least 6 mice were tested in each group. *p<0.05 vs. vessels from PBS-treated apoE^-/- mice. ^#p<0.05 vs. vessels from Ang II-treated apoE^-/- mice.

We also examined the activity of NADPH oxidase after 1 week of Ang II infusion, at which time few adhesive mononuclear cells were observed in the intima (data not shown), and found no significant difference between the two groups of mice (Supplemental Figure 4B). These findings suggest that vascular-targeted overexpression of AT₂ exerts its anti-oxidative action by attenuating the accumulation of superoxide-producing mononuclear leukocytes rather than by a direct effect on vascular NADPH oxidase activity.

Discussion

This is the first study to indicate that targeted overexpression of AT₂ in VSMCs significantly attenuates atherosclerotic lesion development in an endothelial kinin/NO-dependent manner. We previously demonstrated that AT₂ stimulation in VSMCs promotes intracellular acidosis by blocking the amiloride-sensitive Na⁺/H⁺ exchanger, leading to activation of kininogenases followed by bradykinin release, which causes endothelial NO-dependent vasodilation in mice.¹⁶ Accumulating evidence supports the notion that the AT₂ stimulates a vasodilator signaling cascade that includes bradykinin, NO, and guanosine cyclic 3’, 5’-monophosphate.²⁶ The present study using AT₂-Tg/apoE^-/- mice provides new evidence that the AT₂ bradykinin NO system exerts its anti-atherogenic action by attenuating the increased VCAM-1 expression and monocyte/macrophage accumulation, both of which are essentially involved in the early stage of atherosclerosis development. Furthermore, the protective effect of AT₂ on oxidative stress was mostly exerted through reduced accumulation of superoxide-producing mononuclear leukocytes rather than by a direct effect on vascular NADPH oxidase activity. These findings provide new insight into the AT₂-mediated anti-atherogenic actions and support the possibility that direct AT₂ activation could be a potential therapeutic strategy for preventing the development of atherosclerotic cardiovascular diseases.

AT₂ is substantially expressed by endothelial cells of the microvasculature,^27,28 whereas its expression in larger arteries such as the thoracic aorta was barely detectable by a conventional binding assay or immunohistochemistry.^16,28 However, an autoradiography study revealed that a low level of aortic AT₂ expression was detected in the cell layer at the medial–adventitial border rather than the medial–endothelial wall.²⁹ Similarly, using a newly developed polyclonal anti-AT₂ antiserum, Utsunomiya et al.¹⁴ reported that AT₂ expression was observed only in the aortic muscular medial layer and not in the endothelium. Furthermore, both AT₁ and AT₂ expression were up-regulated in the aortas of apoE^-/- mice fed an atherogenic diet.^9,15 Sales et al. showed that the mRNA expression of the AT₂ was threefold higher in apoE^-/- mice fed an atherogenic diet compared with those on standard chow diet.¹⁵ Iwai et al. also observed that the AT₂ was expressed in both the lesion and the aortic medial wall in apoE^-/- mice on a high-cholesterol diet.⁹ The previous in vitro studies investigating the biological effects of AT₂ in VSMCs and endothelial cells showed the discordant results, which is probably due to the difference in animal species and cell lines to be tested. The biological relevance of these results should be assessed in vivo experiments. From this point of view, AT₂-Tg/apoE-/- mice in which AT₂ is overexpressed in aortic VSMCs might be a suitable model for investing the vascular action of aortic AT₂ in atherogenesis. Our findings suggest that AT₂ receptor in VSMCs plays a critical role in atherogenesis, especially in hypercholesterolemia which elicits re-expression of the aortic AT₂ in the medial cell layer including VSMCs.

Studies investigating the effect of AT₂ on the development of atherosclerosis showed conflicting results. AT₂ exerted an anti-atherogenic effect in AT₂/apoE double-deficient mice,⁹ whereas no such action was observed in AT₂/LDL double-deficient mice.¹⁵ These discrepancies most likely resulted from the different stages of atherosclerosis investigated. For example, the atherosclerotic lesion areas in AT₂/apoE double-deficient mice were estimated after 10 weeks on a high-cholesterol diet, corresponding to the early stage of atherosclerosis, whereas the AT₂/LDL double-deficient mice were analyzed at 5 months of age, at which point the plaque evolution was further advanced. Similarly, Daugherty et al. demonstrated that AT₂ antagonist PD123319 treatment exaggerated the Ang II-induced atherosclerotic lesion area in 2-month-old apoE^-/- mice, whereas no such effect was observed at the age of 11 months.⁷

Long-term treatment with a Western diet has been shown to impair endothelial function in apoE^-/- mice.^30,31 Considering that Ang II-induced Ca²⁺-dependent NOS activity was enhanced in AT₂-Tg/apoE^-/- mice, the AT₂-mediated anti-atherogenic effect is likely to be exerted in the early stage of atherosclerosis, during which endothelial function was preserved. Therefore, it is of great importance that the tissue-specific effect and time-dependent action of vascular AT₂ is carefully considered for estimating AT₂-mediated anti-atherogenic actions in the multifactorial process of atherosclerosis.

Reactive oxygen species (ROS) and NO are crucial for the development of atherosclerosis.³² AT₂ has been shown to both increase NO production¹⁶ and inhibit ROS production⁹; however, the precise mechanism for AT₂-mediated modulation of oxidative stress remains to be determined. Iwai et al.⁹ demonstrated that the AT₂ blocker PD123319 augmented Ang II-induced NADPH oxidase activity in rat fetal VSMCs, suggesting that the anti-atherogenic effect of AT₂ occurs, in part, through the inhibitory effect on NADPH oxidase activation, a dominant source of oxidative stress in the vasculature.²⁵ However, the present study did not show an inhibitory effect of AT₂ on NADPH oxidase activity (Supplemental Figure 4B). Iwai et al. stimulated fetal VSMCs with 10^-7 M Ang II and observed a significant increase in NADPH oxidase activity,⁹ while the present study showed that a subpressor dose of Ang II did not increase NADPH oxidase activity (Supplemental Figure 4B), suggesting that AT₂ stimulation was likely to antagonize AT₁-mediated NADPH oxidase activity by directly inhibiting crosstalk of the two receptors. Accordingly, the discrepancy between these two experiments might be attributable to the extent of AT₁-mediated augmentation of NADPH oxidase activity.

The clinical relevance of AT₂ receptor has not been confirmed because of the lack of a selective AT₂ agonist available in clinical practice. Recently, compound 21 (C21), a non-peptide, orally active and selective AT₂ agonist has been developed.¹⁰ Direct AT₂ stimulation by C21 has been reported to exert beneficial effects on cardiac and vascular function in rodents through an increased production of NO and/or suppression of the inflammatory response.^33,34 Rehman et al.³⁴ reported that endothelium-dependent relaxation in isolated rat aortas was enhanced in the C21–losartan combination groups compared with losartan alone, suggesting that C21 treatment increases NO production, ARB treatment inhibits superoxide production, and both lead to the increased NO bioavailability. Considering that the expression of AT₂ receptor is up-regulated in various pathological conditions including post-myocardial infarction, heart failure, and arterial injury,^11–13 the beneficial effect of AT₂ is expected to be assessed in the future clinical trials. Our findings strongly support the notion that direct AT₂ stimulation exerts anti-atherogenic action and could be a potential therapeutic strategy for producing a clinically beneficial effect on the cardiovascular system.

Conclusions

AT₂ activation in VSMCs markedly inhibited the initiation of atherosclerotic lesion development in an endothelial bradykinin/NO-dependent manner, and AT₂-mediated modulation of oxidative stress was mostly attributable to the enhanced NO production and inhibited accumulation of superoxide-producing leukocytes. These findings provide novel evidence of vascular AT₂-mediated anti-atherogenic actions and new insight into the mechanism for AT₂-mediated anti-oxidative action, which could resolve the controversial issue regarding the AT₂-mediated effect on atherosclerosis.

Footnotes

Conflict of interest

None declared.

Funding

This work was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Supplementary Material

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Vascular angiotensin II type 2 receptor attenuates atherosclerosis via a kinin/NO-dependent mechanism

Abstract

Introduction:

Materials and methods:

Results:

Conclusions:

Keywords

Introduction

Materials and methods

Animal preparation

Membrane preparation and receptor assay

Hemodynamic analysis

Plasma lipid analysis

Quantitative measurement of atherosclerotic lesions

Immunohistochemistry

In situ detection of superoxide anions

Real-time polymerase chain reaction

Nicotinamide adenine dinucleotide phosphate oxidase activity

Measurement of Ca2+-dependent NO synthase activity

Statistical analysis

Results

Expression of AT2 in the aortas of AT2-Tg/apoE-/- mice

Vascular AT2 activation inhibits atherosclerosis development in apoE-/- mice

Blockade of the bradykinin/NO system by L-NAME or icatibant abolishes AT2-mediated anti-atherogenic actions

Vascular AT2 activation increases Ca2+-dependent NOS activity in apoE-/- mice

Vascular AT2 inhibits Ang II-induced VCAM-1 expression and monocyte/macrophage accumulation

Vascular AT2 attenuates Ang II-induced oxidative stress by inhibiting the accumulation of superoxide-producing mononuclear leukocytes

Discussion

Conclusions

Footnotes

Conflict of interest

Funding

References

Supplementary Material

Measurement of Ca²⁺-dependent NO synthase activity

Expression of AT₂ in the aortas of AT2-Tg/apoE^-/- mice

Vascular AT₂ activation inhibits atherosclerosis development in apoE^-/- mice

Blockade of the bradykinin/NO system by L-NAME or icatibant abolishes AT₂-mediated anti-atherogenic actions

Vascular AT₂ activation increases Ca²⁺-dependent NOS activity in apoE^-/- mice

Vascular AT₂ inhibits Ang II-induced VCAM-1 expression and monocyte/macrophage accumulation

Vascular AT₂ attenuates Ang II-induced oxidative stress by inhibiting the accumulation of superoxide-producing mononuclear leukocytes