Deletion of Angiotensin II Type 2 Receptor Exaggerated Atherosclerosis in Apolipoprotein E–Null Mice
Background— The role of angiotensin II (Ang II) type 2 (AT2) receptor in atherosclerosis was explored with the use of AT2 receptor/apolipoprotein E (ApoE)–double-knockout (AT2/ApoE-DKO) mice, with a focus on oxidative stress.
Methods and Results— After treatment with a high-cholesterol diet (1.25% cholesterol) for 10 weeks, ApoE-knockout (KO) mice developed atherosclerotic lesions in the aorta. In AT2/ApoE-DKO mice receiving a high-cholesterol diet, the atherosclerotic changes were further exaggerated, without significant changes in plasma cholesterol level and blood pressure. In the atherosclerotic lesion, an increase in superoxide production, NADPH oxidase activity, and expression of p47phox was observed. These changes were also greater in AT2/ApoE-DKO mice. An Ang II type 1 (AT1) receptor blocker, valsartan, inhibited atherosclerotic lesion formation, superoxide production, NADPH oxidase activity, and p47phox expression; these inhibitory effects were significantly weaker in AT2/ApoE-KO mice. We further examined the signaling mechanism of the AT2 receptor–mediated antioxidative effect in cultured fetal vascular smooth muscle cells. NADPH oxidase activity and phosphorylation and translocation of p47phox induced by Ang II were inhibited by valsartan but enhanced by an AT2 receptor blocker, PD123319.
Conclusions— These results suggest that AT2 receptor stimulation attenuates atherosclerosis through inhibition of oxidative stress and that the antiatherosclerotic effect of valsartan could be at least partly due to AT2 receptor stimulation by unbound Ang II.
Received November 29, 2004; revision received April 21, 2005; accepted June 13, 2005.
Angiotensin II (Ang II) is a potent regulator of the cardiovascular system and is involved in vascular inflammation, oxidative stress, and cell proliferation, which are critical steps in atherosclerosis.1,2 Results of clinical studies also suggest that blockade of Ang II type 1 (AT1) receptor decreases events related to atherosclerosis.1,2 We have previously demonstrated that atherosclerotic lesions in apolipoprotein E–null (ApoE-knockout [KO]) mice with deletion of the AT1a receptor were significantly attenuated.3 However, the role of the type 2 (AT2) receptor in atherosclerosis is not yet totally clear. Previous reports suggest that AT2 receptor expression is decreased in most tissues after birth but increased again in certain pathological conditions.4 Stimulation of this increased AT2 receptor appears to regulate neointimal formation, cell proliferation, and inflammation in vascular injury and myocardial infarction.5–7 We have previously demonstrated that beneficial organ-protective effects of AT1 receptor blockers are at least partly due to AT2 receptor stimulation.5,8 AT2 receptor stimulation interacts with AT1 receptor stimulation at intracellular signaling molecules, such as through activation of phosphatases.9 AT1 receptor stimulation is known to activate NADPH oxidase, thereby inducing oxidative stress,3,10 whereas the role of the AT2 receptor in regulation of oxidative stress is poorly known.
ApoE-KO mice are widely used as an animal model of atherosclerosis. Treatment with a high-cholesterol diet induces atherosclerotic lesions predominantly in the aorta. Previous reports have shown that oxidative stress plays an important role in endothelial cell dysfunction and atherosclerotic formation.11 Activation of NADPH oxidase produces superoxide, which then causes oxidation of membrane lipids and activates protein kinases.12–14
In the present study we investigated the possible role of the AT2 receptor in atherosclerosis using AT2 receptor/ApoE–double-knockout (AT2/ApoE-DKO) mice. The results suggested that AT2 receptor stimulation might be involved in atherosclerotic changes partly through oxidative stress and that the antiatherosclerotic effect of valsartan could be at least partly due to AT2 receptor stimulation by unbound Ang II.
Animals and Treatment
To generate AT2/ApoE-DKO mice (backcrossed >10 times), ApoE-KO (B6.129P2-Apoetm1Unc, Jackson Laboratory, Bar Harbor, Maine, backcrossed 10 times) mice and AT2-KO mice (based on C57BL/6J strain)5 were bred to yield mice heterozygous at the ApoE locus and heterozygous (female) and hemizygous (male) at the AT2 receptor loci because the AT2 receptor is located on the X chromosome. These mice were crossed and intercrossed to yield AT2−/− ApoE−/− mice. The Animal Studies Committee of Ehime University approved the following experimental protocol. The animals were housed in a room with a 12-hour light/dark cycle, and temperature was maintained at 25°C. Adult male mice were given a standard diet (MF, Oriental Yeast) or high-cholesterol diet (1.25% cholesterol, 10% coconut oil in MF) for 10 weeks from 6 weeks of age and water ad libitum. Valsartan (provided by Novartis Pharma AG) (1 mg/kg per day), an AT1 receptor–selective AT1 receptor blocker, was administered with an osmotic minipump (Alzet model 1002, DURECT Corporation) implanted intraperitoneally as described previously.5 Plasma cholesterol level was measured by the cholesterol oxidase method (Cholesterol E-test, WAKO Chemical Industries, Ltd). Blood pressure was measured by the indirect tail-cuff method with a blood pressure monitor (MK-1030, Muromachi Kikai Co Ltd).
Determination of Atherosclerotic Lesion Size
The mice were killed at the age of 16 weeks, and the atherosclerotic lesions were analyzed as described previously.3,15,16 The aorta was perfused with PBS by cardiac puncture, and then the aorta was dissected and cleaned of adherent connective tissue. The proximal portion of the thoracic aorta up to the aortic origin (aortic arch) was isolated, cleaned of the adherent connective tissue, embedded in Shandon M-1 Embedding Matrix (Thermo Electron Corporation), and stored at −80°C until use or fixed in 10% neutral buffered formalin overnight.
The area of atherosclerotic lesions in the proximal aorta and the lipid area in the area of aortic wall and the atherosclerotic lesions were determined by means of cross sections taken intermittently throughout 2 to 3 mm of the aortic arch with oil red O staining and counterstaining with hematoxylin. Quantitative analysis was performed with Densitograph Imaging Software (ATTO Corporation). Lesions in the descending aorta were also analyzed. The excised aorta (from aortic arch to iliac bifurcation) was dissected free from surrounding tissues and opened longitudinally. Atherosclerotic lesion area was quantified by analyzing the open luminal surface of oil red O–stained aorta with Densitograph Imaging Software. The amount of lesion formation in each animal was measured as the percentage of lesion area per total area of the aortic endothelial surface. The mean value of 5 sections was taken as the value for each animal.
Immunohistochemical staining of AT1 and AT2 receptors was performed with antibodies (Santa Cruz Biotechnology, Inc) and detected with diaminobenzidine.
Frozen, enzymatically intact, 10-μm-thick sections of cross sections of the proximal aorta were incubated with dihydroethidium (10 μmol/L) in PBS for 30 minutes at 37°C in a humidified chamber protected from light.3,17 Dihydroethidium is oxidized on reaction with superoxide to ethidium, which binds to DNA in the nucleus and fluoresces red. For ethidium detection, a 543-nm argon laser combined with a 500- to 550-nm bandpass filter was used. The intensity of fluorescence was normalized by staining the control section from the same aorta in each assay.
Immunofluorescence was assessed with the use of fresh-frozen sections. Sections were incubated with anti-p47phox antibody, washed, and incubated with biotin-labeled secondary antibodies, then incubated with Cy3-labeled streptavidin. Serial sections treated with secondary antibodies alone did not show specific staining. Samples were examined with a Zeiss Axioskop microscope equipped with a computer-based imaging system.3 The intensity of fluorescence was expressed as pixels for fluorescence and normalized by staining the control section from the same aorta in each assay.
Cell Culture and Treatment
Fetal vascular smooth muscle cells (VSMCs) were prepared from the thoracic aorta of Sprague-Dawley rat fetuses (embryonic day 20) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies Inc) supplemented with 10% fetal bovine serum (FBS) as previously described.18 All the experiments were performed with the use of cells at the second passage. Rat fetal VSMCs were grown in 10% FBS-DMEM to a subconfluent state and incubated for an additional 24 hours in serum-free DMEM to reach a quiescent state. In some experiments, adult VSMCs were isolated from adult Sprague-Dawley rat thoracic aorta (9 weeks of age; Clea Japan Inc) as previously described, exclusively expressing the AT1 receptor but not AT2 receptor.19 Adult VSMCs at passage 3 to 8 were used for the experiments. Subconfluent adult VSMCs were serum-starved for 48 hours to induce a quiescent state before the experiments.
The cells were incubated with Ang II (10−7 mol/L) for the indicated times. In some experiments, VSMC were treated with an AT1 receptor blocker, valsartan (10−5 mol/L) (provided by Novartis Pharma AG, Basel, Switzerland), or an AT2 receptor blocker, PD123319 (10−5 mol/L) (Sigma Chemical Co), for 15 minutes and further incubated with Ang II in the presence of valsartan or PD123319. AT1 and AT2 receptor binding was measured as previously described.18
Measurement of NADPH Oxidase Activity in Aortic Tissue and Cultured Cell
An aortic arch sample was rapidly removed with the intact endothelium, homogenized in 500 μL ice-cold Tris-sucrose buffer (10 mmol/L Tris, pH 7.1, 340 mmol/L sucrose, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin) and incubated for 30 minutes. Samples were centrifuged (15 000g, 10 minutes, 4°C), and the supernatant (20 μg protein) was added to reaction buffer (78 μmol/L cytochrome c [Sigma-Aldrich], 100 μmol/L NADPH [Sigma Chemical Co], with or without 1000 U/mL superoxide dismutase [Sigma Chemical Co]), and then incubated at 37°C for 60 minutes. NADPH oxidase activity was quantified from the absorbance with or without superoxide dismutase as previously described.20
NADPH oxidase activity in cultured cell sample was measured as previously described with slight modification.21 Cell samples were centrifuged, and the pellet was resuspended in ice-cold Tris-sucrose buffer. NADPH oxidase activity was measured in a luminescence assay with 5 μmol/L lucigenin as the electron acceptor, with the use of 100 μmol/L NADPH as the substrate. Chemiluminescence was monitored with a luminometer (AB-2200, ATTO Corporation) for 5 minutes after the addition of cell extract.
Membrane Preparations From Fetal VSMCs
The membrane fraction of fetal VSMCs was isolated by the method of differential centrifugation based on standard protocols, as previously described.22 The cell homogenate was lightly centrifuged to remove unbroken parts and the nuclei-enriched fraction and then centrifuged for 60 minutes at 100 000g to obtain the membrane fraction. The pellets were suspended in homogenization buffer, and protein concentration was determined before immunoblotting.
Immunoprecipitation and Immunoblotting
Equal amounts of protein were incubated at 4°C with anti-p47phox antibodies (Santa Cruz Biotechnology) for 3 hours with constant agitation and then further incubated with protein G-Sepharose 4 Fast Flow (Amersham Pharmacia Biotech) for 1 hour. For immunoblotting, the membrane fraction, whole-cell lysate, and immunoprecipitates were subjected to SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated with anti-phosphoserine specific monoclonal antibodies (Sigma Chemical Co), anti-Rac1 antibodies (Upstate Biotechnology Inc), or anti-p47phox antibodies and then visualized with an ECL detection kit (Amersham Pharmacia Biotech).
Values are expressed as mean±SE in the text and figures. The data were analyzed by ANOVA. If a statistically significant effect was found, Newman-Keuls test was performed to detect the difference between the groups. A value of P<0.05 was considered statistically significant.
Atherosclerotic Lesion Formation in AT2/ApoE-DKO Mice
Treatment with a high-cholesterol diet for 10 weeks markedly increased plasma cholesterol level but did not significantly affect blood pressure in ApoE-KO mice (Table). In AT2/ApoE-DKO mice, blood pressure and plasma cholesterol level were not significantly different from those in ApoE-KO mice, both before and after treatment with high-cholesterol diet (Table).
After treatment with high-cholesterol diet for 10 weeks, atherosclerotic lesions and lipid accumulation were observed in the proximal aorta of ApoE-KO mice (Figure 1A and 1B) as well as in descending aorta (Figure 1C). In our experimental schedule, ApoE-KO mice treated with normal standard diet did not show apparent atherosclerotic changes. In the atherosclerotic lesions in ApoE-KO mice, expression of the AT1 and AT2 receptors determined by immunohistochemical staining was increased not only in the lesion but also in the aortic wall (Figure 2). Treatment with valsartan did not seem to change AT1 and AT2 receptor expression apparently (Figure 2). AT1 receptor expression in AT2/ApoE-DKO mice was increased to a degree similar to that in ApoE-KO mice (data not shown). In AT2/ApoE-DKO mice receiving a normal standard diet, the atherosclerotic area and lipid accumulation in the proximal aorta were not significantly different from those in ApoE-KO mice. However, after mice received a high-cholesterol diet, the atherosclerotic area and lipid accumulation in the proximal aorta were further enhanced compared with those in ApoE-KO mice (Figure 1).
Changes in Oxidative Stress in Atherosclerotic Lesion of AT2/ApoE-DKO Mice
Figure 3 shows the in situ detection of superoxide in the proximal aorta with the use of dihydroethidium. Superoxide production was increased in the atherosclerotic aorta of ApoE-KO mice receiving high-cholesterol diet compared with those receiving a normal diet. This increase in superoxide production was further exaggerated in AT2/ApoE-DKO mice, not only in the atherosclerotic lesion but also in smooth muscle cells (Figure 3). NADPH oxidase activity and expression of the NADPH oxidase subunit, p47phox, were also increased in the atherosclerotic aorta (Figures 4 and 5⇓). In parallel with the changes in superoxide production, NADPH oxidase activity and expression of p47phox were further enhanced in the lesion area and aortic wall of AT2/ApoE-DKO mice.
Effect of Valsartan on Atherosclerotic Changes in AT2/ApoE-DKO Mice
Treatment with an AT1 receptor blocker, valsartan, at a dose of 1 mg/kg per day inhibited atherosclerotic lesion formation and lipid accumulation as well as superoxide production, NADPH oxidase activity, and NADPH oxidase expression in the aorta of ApoE-KO mice given a high-cholesterol diet (Figure 1 and Figures 3 to 5⇑⇑). However, these inhibitory effects of valsartan on atherosclerosis were significantly weaker in AT2/ApoE-DKO mice (60.0% versus 38.6% inhibition of proximal lesion area, 55.3% versus 19.8% inhibition of proximal lipid deposition, and 91.8% versus 53.5% inhibition of plaque area in descending aorta for ApoE-KO and AT2/ApoE-DKO mice, respectively; P<0.05; Figure 1B and 1C). Similar attenuation of the inhibitory action of valsartan in AT2/ApoE-DKO mice was observed for superoxide production (81.2% versus 53.7% inhibition for ApoE-KO and AT2/ApoE-DKO mice, respectively; P<0.05), NADPH oxidase activation (35.8% versus 12.2% inhibition for ApoE-KO and AT2/ApoE-DKO mice, respectively; P<0.05), and p47phox expression (68.4% versus 47.4% inhibition for ApoE-KO and AT2/ApoE-DKO mice, respectively; P<0.05) in Figures 3B, 4, and 5⇑⇑B.
Effect of AT2 Receptor Stimulation on NADPH Oxidase Activity in Fetal VSMCs
We next examined the signaling mechanism of action of AT2 receptor stimulation on NADPH oxidase activity using rat fetal VSMCs, which expressed both AT1 and AT2 receptors (AT1 receptor, 11.21±0.42 fmol/106 cells; AT2 receptor, 6.58±1.09 fmol/106 cells), as previously described.18 Ang II increased NADPH oxidase activity in fetal VSMCs after 3 hours of stimulation. An AT1 receptor blocker, valsartan, inhibited the Ang II–mediated increase in NADPH oxidase activity (Figure 6). However, NADPH oxidase activity induced by Ang II was further increased in the presence of PD123319 after 30 minutes. Neither valsartan nor PD123319 alone influenced NADPH oxidase activity (Figure 6A). Moreover, when the cells were pretreated with valsartan, PD123319 did not enhance NADPH oxidase activity at 30 minutes after Ang II stimulation (Figure 6B; Ang II, 119.3±3.2%; Ang II+valsartan, 100.5±3.2%; Ang II+valsartan+PD123319, 101.2±2.1%, expressed as percentages from basal level; n=5). The Ang II receptor subtype detected by receptor binding assay was not significantly changed in these experimental conditions (data not shown).
On the other hand, the same dose of valsartan completely inhibited the effect of Ang II on NADPH oxidase activation even in adult VSMCs (Figure 6B), which dominantly expressed AT1 receptor (9.10±0.59 fmol/106 cells) and AT2 receptor (0.08±0.02 fmol/106 cells), as previously reported.19
Effect of AT2 Receptor Stimulation on p47phox Subunit of NADPH Oxidase, Rac1, and Akt in Fetal VSMCs
The effects of AT1 or AT2 receptor stimulation on serine phosphorylation of the p47phox subunit of NADPH oxidase was examined in fetal VSMCs (Figure 7A). Ang II stimulation for 10 minutes significantly increased serine phosphorylation of p47phox. Valsartan decreased Ang II–mediated serine phosphorylation of p47phox, whereas treatment with PD123319 further increased it (Figure 7A). Moreover, Ang II stimulation significantly increased translocation of p47phox and Rac1 to the plasma membrane fraction of fetal VSMCs. This translocation was attenuated by the addition of valsartan but was further increased by the addition of PD123319 (Figure 7B and 7C). Ang II–mediated Akt phosphorylation in fetal VSMCs was also inhibited by valsartan but was enhanced by PD123319 (Figure 7D).
To explore the role of AT2 receptor stimulation in the pathogenesis of atherosclerosis using ApoE-KO mice, we generated a new strain in which the AT2 receptor is deleted (AT2/ApoE-DKO mice). The present study showed that atherosclerotic changes were aggravated in AT2/ApoE-DKO mice compared with ApoE-single-KO mice. The atherosclerotic change was associated with an increase in oxidative stress determined by superoxide production, NADPH oxidase activity, and NADPH oxidase subunit expression. These changes were also further enhanced in AT2/ApoE-DKO mice. In in vitro experiments in which fetal VSMCs were used, AT2 receptor stimulation inhibited NADPH oxidase activity and expression and/or translocation of subunits of NADPH oxidase. These results indicate that AT2 receptor stimulation plays an important role in atherosclerotic lesion formation through regulation of oxidative stress.
It is reported that AT1 receptor stimulation is involved in the development of atherosclerosis in ApoE-KO mice. Indeed, it was previously reported that atherosclerotic changes were significantly attenuated in AT1a/ApoE-KO mice.3,23 The effects of AT1 receptor stimulation appear to be mediated by inflammatory responses, oxidative stress, and cell proliferation.3,5 In contrast, previous results suggest that AT2 receptor stimulation exerts antiinflammatory and antiproliferative effects on the vasculature.4,5,7 However, the effect of AT2 receptor stimulation on oxidative stress is still an enigma. In the present study in which AT2/ApoE-DKO mice were used, it was indicated that a lack of AT2 receptor induced significant enlargement of atherosclerotic lesions after treatment with a high-cholesterol diet. This enlargement in AT2/ApoE-DKO mice did not depend on changes in systemic blood pressure and plasma cholesterol level, suggesting that AT2 receptor stimulation inhibits atherosclerotic changes in ApoE-KO mice. This effect of the AT2 receptor stimulation seems to be mediated at least in part by oxidative stress as well as inflammatory response (Figures 3 to 5⇑⇑).3,5,8 In contrast, it has very recently been reported that AT2 receptor deficiency had no effect on atherosclerosis lesion area in LDL receptor–deficient mice.24 This apparently different result for the role of AT2 receptor from that in the present report might be due to the difference in animal model, although future study is necessary to clarify the detailed mechanism of AT2 receptor function.
Oxidative stress, including production of reactive oxygen species, plays an important role in the pathogenesis of atherosclerosis.25 Previous studies suggest that NADPH oxidase is a key enzyme in superoxide production and that the enzyme activity is regulated by expression of its subunit.26 It is also reported that AT1 receptor stimulation activated reactive oxygen species production in vascular cells in vitro as well as in vivo.26,27 In the mouse microvascular endothelial NADH/NADPH system, Ang II stimulated NADH/NADPH oxidase activity through serine phosphorylation of p47phox and its enhanced binding to p22phox.28 In the present study superoxide production, NADPH oxidase activity, and expression of an NADPH oxidase subunit, p47phox, were markedly increased in atherosclerotic lesions (Figures 3 to 5⇑⇑). This increase in oxidative stress was located not only in the lesion but also in aortic smooth muscle cells. The increase in superoxide production, NADPH oxidase activity, and p47phox expression was further enhanced in AT2/ApoE-DKO mice, similar to the change in atherosclerotic lesion area (Figures 3 to 5⇑⇑). These results suggest that the antiatherosclerotic effects of AT2 receptor stimulation are mediated at least partly through modulation of oxidative stress.
In cultured fetal VSMCs, blockade of the AT2 receptor by PD123319 significantly enhanced the Ang II–induced increase in NADPH oxidase activity, whereas an AT1 receptor blocker, valsartan, inhibited it. Moreover, PD123319 did not enhance NADPH oxidase activity after pretreatment of cells with valsartan. Because PD123319 or valsartan alone did not affect basal NADPH activity, these results indicate that AT2 receptor stimulation antagonized AT1 receptor–mediated NADPH oxidase activation. AT1 receptor stimulation seems to be a prerequisite for the AT2 receptor to exert its inhibitory effect on NADPH oxidase activation. It is well known that serine phosphorylation of the p47phox subunit of NADPH oxidase plays a critical role in the activation of NADPH oxidase.28,29 In our experiments the regulation of NADPH oxidase activity appeared to be mediated by phosphorylation of p47phox and translocation of Rac1 to the plasma membrane (Figure 7). The mechanism of action of AT2 receptor stimulation on NADPH oxidase activity includes, at least in part, the inhibition of the activation and translocation of NADPH oxidase subunits. The detailed pathway of intracellular signaling after NADPH oxidase mediated by AT2 receptor has not yet been clarified. However, blockade of the AT2 receptor increased the phosphorylation of Akt in fetal VSMCs. Previous reports also indicate the important role of Akt in the action of oxidative stress.30 These results suggest that Akt activation mediates intracellular signaling in the aorta to produce atherosclerosis and that AT2 receptor stimulation exerts an inhibitory action by regulation of oxidative stress, at least in part, through Akt activation. Taken together, our results provide novel insights into the pathogenesis of atherosclerosis regulated by distinct Ang II receptor subtypes. More detailed analysis of the AT2 receptor–mediated antiatherosclerotic effect would contribute to the development of a new rationale for therapeutic concepts.
This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan, Cardiovascular Research Foundation, Mitsubishi Pharma Research Foundation, Takeda Science Foundation, and Novartis Foundation of Gerontological Research.
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The detailed mechanism of atherosclerosis formation and roles of angiotensin II (Ang II) receptor subtypes are not yet clear. Results of clinical studies also suggest that blockade of Ang II type 1 (AT1) receptor decreases events related to atherosclerosis. The results in our previous reports suggest that Ang II type 2 (AT2) receptor stimulation induces beneficial effects in cardiovascular remodeling, which could be expected with an AT1 receptor blocker. In the present study we examined the role of AT2 receptor stimulation in atherosclerosis formation. Apolipoprotein E–deficient (ApoE-knockout [KO]) mice treated with a high-cholesterol diet are commonly used as an animal model of atherosclerosis. For this study we generated double gene–knockout mice, which lack both AT2 receptor and ApoE (AT2/ApoE-DKO mice). Compared with ApoE-KO mice, less enhancement of atherosclerotic formation and the weaker antiatherosclerotic effect of an AT1 receptor blocker, valsartan, in AT2/ApoE-DKO mice indicated that the inhibitory action of AT2 receptor stimulation on atherosclerosis plays a role and the involvement of AT2 receptor stimulation is associated with the effects of AT1 receptor blockers. Because the cardiovascular system also expresses AT2 receptor in humans, especially in diseased states such as vascular injury and atherosclerosis, our results suggest the therapeutic beneficial effects of AT2 receptor stimulation on atherosclerosis. Our results propose a possibility of therapeutic use of AT2 receptor stimulant for patients with atherosclerosis. Although a selective AT2 receptor agonist has not yet been developed for the clinical setting, the development of a stable AT2 receptor agonist might provide us with a new therapeutic tool for cardiovascular disease.