Attenuation of Inflammatory Vascular Remodeling by Angiotensin II Type 1 Receptor–Associated Protein
To explore the role of angiotensin II Type 1 receptor–associated protein (ATRAP) in vascular remodeling, we developed transgenic mice for mouse ATRAP cDNA and examined remodeling after inflammatory vascular injury induced by polyethylene cuff placement. In ATRAP transgenic (ATRAP-Tg) mice, ATRAP mRNA was increased 3- to 4-fold in the heart, aorta, and femoral artery. ATRAP-Tg mice showed no significant change in body weight, systolic blood pressure, heart rate, and heart/body weight ratio. However, cell proliferation and neointimal formation in the injured artery were attenuated in ATRAP-Tg mice. The increase in NADPH oxidase activity and the expression of p22phox, a reduced nicotinamide-adenine dinucleotide/reduced nicotinamide-adenine dinucleotide phosphate oxidase subunit, after cuff placement was also attenuated in ATRAP-Tg mice. Moreover, activation of extracellular signal–regulated kinase, signal transducer and activator of transcription 1, and signal transducer and activator of transcription 3 after cuff placement was significantly reduced in ATRAP-Tg mice. Pressor response and cardiac hypertrophy induced by angiotensin II infusion and pressure overload were also attenuated in ATRAP-Tg mice. These results suggest that ATRAP plays an important role in vascular remodeling as a negative regulator.
The cardiovascular actions of angiotensin II (Ang II) are mainly mediated by the Ang II type 1 (AT1) receptor. Previous reports indicate that the intracellular carboxyl-terminal tail of the receptor plays an important role in activation of receptor-coupled G protein and internalization of the AT1 receptor.1–6 We cloned a novel AT1 receptor-associated protein (ATRAP) using a yeast 2-hybrid screening system.7 ATRAP has 3 transmembrane domains and interacts with the intracellular carboxyl-terminal domain of the AT1 receptor, but it does not interact with the AT2 receptor, m3 muscarinic receptor, bradykinin B2 receptor, endothelin ETB receptor, or β2-adrenergic receptor. It is reported that ATRAP modulates AT1 receptor function in COS-7 cells, human embryonic kidney 293 cells, and cultured mouse cardiomyocytes. Overexpression of ATRAP significantly decreases the number of AT1 receptors on the cell surface and also decreases the degree of p38 mitogen-activated protein kinase phosphorylation, activity of the c-fos promoter, and protein synthesis on Ang II treatment.7–9 We also reported that overexpression of ATRAP in cultured vascular smooth muscle cells (VSMCs) enhanced internalization of the AT1 receptor and attenuated DNA synthesis and activation of extracellular signal-regulated kinase (ERK), Akt, and signal transducer and activator of transcription (STAT) induced by Ang II.10
Polyethylene cuff placement around the femoral artery induces inflammatory vascular injury and remodeling responses accompanied by an increase in AT1 and AT2 receptor expression. VSMC proliferation, neointimal formation, inflammatory response, and oxidative stress in vascular injury were significantly attenuated in AT1 a receptor-deficient mice.11–13 Moreover, administration of an AT1 receptor blocker, valsartan, also decreased vascular remodeling. These results strongly suggest that ATRAP may act as an important regulator of VSMC proliferation and vascular remodeling. In the present study, we prepared transgenic mice for ATRAP and examined its role in inflammatory vascular injury induced by polyethylene cuff placement around the femoral artery.
Transgene Constructs and Screening of Transgenic Mice
The hybrid cytomegalovirus enhancer/chicken β-actin (CAG) promoter and a mouse ATRAP cDNA were subcloned into the plasmid pCAG-GS. The plasmid containing the CAG promoter and ATRAP cDNA was microinjected into the pronuclei of fertilized mouse embryos at the single-cell stage to generate transgenic mice (C57BL/6 strain). Transgenic mice were identified by PCR using 5′-ATGGAGCTGCCTGCCGTGAA-3′ as the forward primer and 5′-GTTCACGGTGCATGTGGTAG-3′ as the reverse primer.
Adult male ATRAP transgenic (ATRAP-Tg) mice (10 to 12 weeks of age) and wild-type mice (C57BL/6J) were used in this study. The mice were housed in a room in which lighting was controlled (12 hours on and 12 hours off), and room temperature was kept at 25°C. They were given a standard diet (MF, Oriental Yeast Co, Ltd) and water ad libitum. The experimental protocol was approved by the Animal Studies Committee of Ehime University. Inflammatory vascular injury was induced by polyethylene cuff placement around the femoral artery under anesthesia with intraperitoneal injection of ketamine (70 mg/kg) and xylazine (4 mg/kg) according to methods described previously,11–14 and morphometric analysis to measure neointimal area was performed as described previously.11–13 The heart/body weight ratio was calculated as whole heart weight (milligrams) divided by body weight (grams). Blood pressure and heart rate were measured under consciousness by the indirect tail- cuff method with a blood pressure monitor (MK-1030, Muromachi Kikai Co, Ltd). The mice were held in a case where temperature was kept at 37°C.
Infusion of Ang II and Aortic Banding
Ang II was infused intraperitoneally at a dose of 1 μg/kg per minute for 2 weeks using an osmotic minipump. The aortic banding was performed under anesthesia with intraperitoneal injection of ketamine (70 mg/kg) and xylazine (4 mg/kg) according to the method described previously.15,16 After 4 weeks, the hearts were excised, weighed, and heart/body weight ratio was expressed as heart weight (milligrams) versus body weight (grams) as described previously.15,16
Rabbit polyclonal antibody against the epitope mapped at the C-terminus of ATRAP (CPFASLENKGQAAPRG) was prepared by the Peptide Institute, Inc. Anti-p22phox antibody was purchased from Santa Cruz Biotechnology Inc.13 Formalin-fixed, paraffin-embedded sections were prepared using femoral artery at 7 days after cuff placement. Proliferating cell nuclear antigen (PCNA) was stained with anti-PCNA antibody (Novocastra Laboratories, Ltd.) using an M.O.M. immunodetection kit (Vector Laboratories, Inc).11,13 ATRAP and p22phox were stained using biotin-labeled secondary antibodies and Cy3-labeled streptavidin as described previously.11,12 Paraffin-embedded sections were incubated with anti-p22phox and ATRAP 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.13
Western Blot Analysis
Total proteins were prepared from pooled arteries (n=3, each pool contained 4 arteries from 4 mice) at 7 days after cuff placement. Phosphorylation of ERK, STAT1, and STAT3 was detected by Western blot using specific antibodies as described previously.13,17
NADPH Oxidase Activity
A tissue protein sample was prepared from the femoral artery at 7 days after cuff placement by homogenization in 500 μL of ice-cold Tris-sucrose buffer. Reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase activity was quantified by cytochrome c method from the absorbance with or without superoxide dismutase, as described previously.18,19
Total RNA was extracted from the femoral arteries (n=3, each pool contained 4 arteries from 4 mice). Real-time quantitative RT-PCR was performed with Premix Ex Taq (Takara Bio Inc). PCR primers for ATRAP were the same as for genotyping; for the AT1 receptor they were 5′-GTTCCTGCTCACGTGTCTCA-3′ (forward) and 5′-CATCAGCCAGATGATGATGC-3′ (reverse); for the AT2 receptor they were 5′-CCTGCATGAGTGTCGATAGGT-3′ (forward) and 5′-CCAGCAGACCACTGAGCATA-3′ (reverse); and for GAPDH they were 5′-ATGTAGGCCATGAGGTCCAC-3′ (forward) and 5′-TGCGACTTCAACAGCAACTC-3′ (reverse).
The data were analyzed by 1-way ANOVA. If a statistically significant effect was found, post hoc analysis by Bonferroni’s test was performed to detect the difference between the groups. A value of P<0.05 was considered statistically significant.
Expression of ATRAP in Injured Artery After Cuff Placement in Wild-Type Mice
Expression of ATRAP in the femoral artery was examined by real-time RT-PCR and immunohistochemical staining. Figure 1A shows immunostaining of ATRAP in the femoral artery at 7 days after cuff placement or sham operation. ATRAP was observed mainly in the media of the intact artery. ATRAP was transiently decreased in the media at 7 days after cuff placement. This decrease in ATRAP after cuff placement was accompanied by a decrease in mRNA (Figure 1B). The decrease in ATRAP protein and mRNA had recovered by 14 days after cuff placement.
Blood Pressure, Heart Rate, Heart/Body Ratio, and ATRAP Expression in ATRAP-Tg Mice
Three founder lines that transmitted the transgene were established by Southern blot (ATRAP-Tg 10, 11, and 15). Because ATRAP-Tg 15 showed the highest expression of ATRAP mRNA (Figure I, available online at http://hyper. ahajournals.org), heterozygous animals of this line were used in the present study. Body size, body weight, and behavior of ATRAP-Tg mice were not different from those of C57BL/6J (wild-type [WT]) mice. As shown in the Table, systolic blood pressure, heart rate, and heart/body weight ratio in ATRAP-Tg mice were not significantly different from those in WT mice. ATRAP expression increased ≈3- to 4-fold in the heart, aorta, and femoral artery of ATRAP-Tg mice (Figure II). Media/lumen ratio of the intact femoral artery in ATRAP-Tg mice also did not differ from that in WT mice (Figure IIIA). Expression of the AT1 receptor in the heart, aorta, and femoral artery was not significantly different between WT and ATRAP-Tg mice (Figure III B).
Effect of ATRAP on VSMC Proliferation and Neointimal Formation After Cuff Placement in ATRAP-Tg Mice
Cuff placement induces proliferation of VSMC and neointimal formation in the femoral artery.12,14,20 Figure 2 shows PCNA labeling index in the media and intima at 7 days after cuff placement in WT and ATRAP-Tg mice. The control (noninjured) levels of PCNA index were not significantly different between WT and ATRAP-Tg groups. However, the increase in PCNA index in the injured artery was suppressed in ATRAP-Tg mice. Related to the change in PCNA index, neointimal formation at 14 days after cuff placement was also attenuated in ATRAP-Tg mice (Figure 3).
Inhibition of Oxidative Stress After Cuff Placement in ATRAP-Tg Mice
The increase in in situ superoxide production in the injured artery at 7 days after cuff placement was significantly attenuated in ATRAP-Tg mice. It is reported that superoxide production is mainly mediated by NADPH oxidase.13,21,22 NADPH oxidase activity in the injured artery was lowered in ATRAP-Tg mice (Figure 4A). Moreover, expression of p22phox, a membrane-associated reduced nicotinamide-adenine dinucleotide/NADPH oxidase subunit, was also attenuated in ATRAP-Tg mice (Figure 4B).
Change in Intracellular Signaling in the Injured Artery of ATRAP-Tg Mice
As reported previously, cuff placement increases phosphorylation of ERK, STAT1, and STAT3 via AT1 receptor stimulation.17,23 The phosphorylation level of these markers was not significantly different between WT and ATRAP-Tg mice (Figure 5). However, the increase in phosphorylation of these markers after cuff placement was suppressed in ATRAP-Tg mice.
Effect of ATRAP on Cardiac Hypertrophy
The degree of cardiomyocyte hypertrophy was evaluated by calculating the ratio of heart weight/body weight. This parameter did not differ between WT and ATRAP-Tg mice before aortic banding (Figure 6). Heart-to-body weight ratio was increased 4 weeks after aortic banding in both mice strains, whereas these parameters were smaller in ATRAP-Tg mice (Figure 6A). Administration of Ang II, at 1 μg/kg per minute for 14 days, increased heart/body weight ratio with the increase in systolic blood pressure, whereas increases in these parameters were less in ATRAP-Tg mice (Figure 6B and 6C).
In the present study, we demonstrated that VSMC proliferation, neointimal formation, and inflammatory response in the injured artery after cuff placement were inhibited in ATRAP-Tg mice compared with those in WT mice. The AT1 receptor is a 7 trans-membrane receptor distributed in most adult tissues and mediates the major action of Ang II. The intracellular carboxy-terminal of the AT1 receptor is capable of binding with intracellular signaling molecules like PLC-γ1, SHP-2, and Jak2.3,4 ATRAP is a novel protein associated with the intracellular carboxy-terminal domain.7 Previous reports suggest that ATRAP accelerates internalization of the AT1 receptor and attenuates the AT1 receptor-mediated response.8–10 Functional analysis of the effects of ATRAP on Ang II-induced AT1 receptor signaling revealed decreases in the generation of inositol lipids, Ang II–stimulated transcriptional activity of the c-fos promoter and STAT, and cell proliferation. A recent study showed that ATRAP interacts with calcium-modulating cyclophilin ligand and decreases Ang II– or calcium-modulating cyclophilin ligand-induced nuclear factor of activated T cell transcriptional activation.24 We reported that overexpression of ATRAP increased internalization of the AT1 receptor in cultured VSMCs. In these cells, an increase in thymidine incorporation and in ERK phosphorylation induced by Ang II was attenuated. These results indicate that ATRAP suppresses AT1 receptor–mediated signaling by stimulating internalization of the receptor. On the other hand, Ang II is involved in vascular remodeling through stimulation of the AT1 receptor. Using an animal model of inflammatory vascular injury induced by polyethylene-cuff placement around the femoral artery, we demonstrated that VSMC proliferation, neointimal formation, and inflammatory response were markedly suppressed by blockade of AT1 receptor–mediated signaling with an ARB or AT1 receptor gene knockout.11,12,17,23 Because the overexpression of ATRAP suppresses AT1 receptor–mediated signaling, it is suggested that the increased ATRAP activity may inhibit vascular remodeling. In fact, ATRAP expression was decreased in the injured artery at 7 days after cuff placement in WT mice, when neointimal formation and inflammation were not yet obvious (Figure 1A and 1B). Therefore, it is possible that change in ATRAP level may affect remodeling of the injured artery.
To examine the function of ATRAP in an in vivo injury model, we developed ATRAP-Tg mice. Expression of ATRAP mRNA in the heart, aorta, and femoral artery in these mice was ≈3- to 4-fold higher than in WT mice (Figure II). However, ATRAP-Tg mice showed no significant change in growth, body weight, heart rate, blood pressure, and heart/body weight ratio (Table). These results indicate that overexpression of ATRAP did not affect basal physiological markers. Expression of AT1 receptor mRNA was not significantly changed in the heart, aorta, and femoral artery of ATRAP-Tg mice. The effect of ATRAP on internalization of the AT1 receptor may appear only when the AT1 receptor is stimulated by Ang II.
ATRAP-Tg mice showed a decrease in vascular remodeling induced by cuff placement (Figure 1A and 1B). Because AT1 receptor stimulation increases inflammation, VSMC proliferation, and oxidative stress,21,25 the results in ATRAP-Tg mice suggest that overexpression of ATRAP inhibits AT1 receptor–mediated responses in vascular injury. We have performed additional studies to examine whether the overexpression of ATRAP could also reduce cardiac hypertrophy related to Ang II and observed that cardiac hypertrophy induced by pressure overload or Ang II infusion and pressor response induced by Ang II infusion were attenuated in ATRAP-Tg mice. These results suggest that overexpression of ATRAP attenuates the cardiac hypertrophy mediated mainly by AT1 receptor stimulation, although it is also possible that the inhibition of cardiac hypertrophy in ATRAP-Tg mice may also be caused by the reduced pressor response, because myocardial hypertrophy can be because of elevated blood pressure, and overexpression of ATRAP attenuated the pressor response to Ang II infusion in our study.
In our vascular injury model, oxidative stress, such as superoxide production and NADPH oxidase activity, was increased after cuff placement.13 This increase in oxidative stress was also suppressed in ATRAP-Tg mice (Figure 4). Moreover, phosphorylation of intracellular signaling molecules like ERK, STAT1, and STAT3, which are stimulated through the AT1 receptor, was attenuated in the injured artery of ATRAP-Tg mice (Figure 5).
Our results suggest that an increase in ATRAP expression in vivo attenuates AT1 receptor–mediated signaling and thereby reduces vascular remodeling. There could be the possibility that increase in expression of ATRAP could be having effects in the intact animal other than AT1 receptor inhibition. However, the detailed mechanisms of the function of ATRAP, for example, the regulation of ATRAP expression, possible ligands for ATRAP, and the regulatory mechanism of the action of ATRAP on phosphorylation and/or dephosphorylation of signaling molecules need to be clarified. The results of the present study suggest that ATRAP may be a novel drug target for the treatment of pathological vascular remodeling.
Sources of Funding
This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan; the Cardiovascular Research Foundation; the Mitsubishi Pharma Research Foundation; Takeda Science Foundation; and the Novartis Foundation of Gerontological Research.
- Received May 16, 2006.
- Revision received June 20, 2006.
- Accepted July 18, 2006.
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