Chapter 3

Animal Models with a Genetic Alteration

of AT2 Expression
Masaki Mogi, Masatsugu Horiuchi
Molecular Cardiovascular Biology and Pharmacology, Ehime University Graduate School of Medicine, Tohon, Shitsukawa, Ehime, Japan

ABBREVIATIONS
α-SMA α-smooth muscle actin
Ang II angiotensin II
ApoE apolipoprotein E
AT1 receptor angiotensin II type 1 receptor
AT2 receptor angiotensin II type 2 receptor
B2R bradykinin subtype 2 receptor
DKO double-knockout
MAP mean arterial pressure
MI myocardial infarction
NO nitric oxide
NOS nitric oxide synthase

INTRODUCTION
Following cloning of the cDNA for the mouse angiotensin II type 2 receptor (AT2 receptor), genetically modified
animals were expected to be generated for functional analysis of AT2 receptor functions. Two kinds of mice with
disruption of the AT2 receptor gene (Agtr2) were reported simultaneously by Hein et al.1 and Ichiki et al.2 In 1995,
Hein et al. first demonstrated that AT2 receptor-disrupted (Agtr2−) mice, which were generated by disruption of the
coding sequence of the Agtr2 gene via insertion of the neomycin resistance gene (neor) into exon 3, develop normally
but have an impaired drinking response to water deprivation as well as a reduction in spontaneous movements.1
Morphological changes in the heart, lung, kidney, adrenal, spleen, brain, aorta, ovary, uterus, pancreas, eye, skeletal
muscle, and blood are not observed in nonmutant, hemizygous and homozygous mutant mice. Ichiki et al. also gener-
ated Agtr2− mice by disruption of the Agtr2 gene via insertion of the neomycin resistance gene (neor) into exon 3.2
Homozygous female mutant mice are healthy and fertile. Likewise, no obvious morphological changes are observed
in the brain (inferior olive and locus coeruleus), heart, lung, kidney, and adrenal of hemizygous male mutant mice.
Generation of Agtr2− mice reveals the functional roles of the AT2 receptor clearly and contributes to progress in re-
search on AT2 receptor functions.

BLOOD PRESSURE
Hein et al. reported that an increased vasopressor response to injection of angiotensin II (Ang II) is found in Agtr2−
mice, although baseline blood pressure is normal.1 In contrast, Ichiki et al. reported that mice lacking the AT2 receptor
show a significant increase in blood pressure and increased sensitivity to the pressor action of Ang II.2 Thus, there is

The Protective Arm of the Renin–Angiotensin System (RAS). http://dx.doi.org/10.1016/B978-0-12-801364-9.00003-1

© 2015 Elsevier Inc. All rights reserved. 17
18  The Protective Arm of the Renin–Angiotensin System (RAS)

a difference in baseline blood pressure and the vasopressor response to injection of Ang II between these two kinds of
Agtr2− mice. Tanaka et al. reported that the increased vascular reactivity to Ang II in Agtr2− mice reported by Ichiki
et al. is at least partly due to increased vascular angiotensin II type 1 receptor (AT1 receptor) expression.3 Therefore,
triple gene-knockout mice deficient in all three Ang II receptors (AT1a, AT1b, and AT2) were generated by intercross-
ing and breeding of single-knockout mice by Walther's group.4 Investigation of triple-knockout mice showed that AT2
receptor deletion increased baseline mean arterial pressure (MAP), whereas mice lacking the AT1a receptor were hypo-
tensive and displayed a reduced heart weight/body weight ratio. Because Ang II failed to alter MAP in triple-knockout
mice, it is likely that no other receptors than AT1a, AT1b, and/or AT2 mediate the pressor effects of Ang II. Central
roles of Ang II receptors in blood pressure regulation have been highlighted. Intracerebroventricular Ang II injection
increased systolic blood pressure in a dose-dependent manner in Agtr2− mice; however, this increase was significantly
greater in Agtr2− mice than in wild-type (C57BL/6) mice.5 Moreover, we generated mice with deletion of both the AT1a
and AT2 receptors (AT1a/AT2-double-knockout (DKO)) by crossing AT1a and AT2-KO mice.5 Intracerebroventricular
Ang II injection-induced blood pressure elevation and water intake were significantly greater in Agtr2− mice, but not
in AT1a/AT2-DKO, indicating that the functional balance of central AT1a and AT2 receptors plays an important role in
the mechanism of hypertension.

CARDIAC DISEASE
Akishita et al. clearly demonstrated that the vasoconstrictor response is exaggerated in Agtr2− mice by evaluating con-
traction of aortic rings by Ang II in vitro6 and that coronary arterial thickening and perivascular fibrosis after aortic
banding are 50% greater in Agtr2− mice than in wild-type mice.7 We reported the potential beneficial effects of AT2
receptor signaling on cardiac remodeling8,9 and heart failure after myocardial infarction (MI)10 in experiments using
Agtr2− mice. Furthermore, chronic loss of the AT2 receptor prevented collagen deposition and caused cardiac rupture
in post-MI models.11 To evaluate the function of the AT2 receptor in local tissues, tissue-specific Agtr2 gene alterations
have been reported since 1998. Masaki et al. demonstrated that mice with cardiac-specific overexpression of the AT2
receptor showed decreased sensitivity to the AT1 receptor-mediated pressor action without any effect on cardiac con-
tractility, which was mainly due to the inhibitory effect of the AT2 receptor on the AT1 receptor-mediated chronotropic
effect,12 and that this mouse strain also shows attenuated perivascular fibrosis by a kinin/NO-dependent mechanism
without the development of cardiomyocyte hypertrophy by Ang II infusion.13 These transgenic mice were generated by
microinjection of α-myosin heavy chain (α-MHC) or α-smooth muscle actin (α-SMA) promoter and AT2 receptor cDNA
into the pronuclei of fertilized embryos. On the other hand, transgenic mice overexpressing AT2 receptors selectively in
ventricular myocytes were generated by Lorell's group, where the AT2 receptor was driven by the myosin light chain-2v
promoter.14 They reported that overexpression of AT2 receptors promotes the development of dilated cardiomyopathy
and heart failure.14 They showed that chronic overexpression of the AT2 receptor has the potential to cause Ca2+- and
pH-dependent contractile dysfunction in ventricular myocytes, as well as loss of the inotropic response to Ang II,15 and
that AT2 receptor signaling modifies the pathological hypertrophic response to aortic banding resulting in chronic pres-
sure overload-induced cardiac hypertrophy.16 In post-MI remodeling, Voros et al. also showed a beneficial effect of the
AT2 receptor using double-gene-alteration mice. They generated these mice using mice with cardiac overexpression of
the AT2 receptor and AT1a-deficient mice and assessed the intricately intertwined connection of these receptors.17 They
showed that genetic deletion of the AT1a receptor is additive to AT2 receptor overexpression, due, at least in part, to blood
pressure lowering. Moreover, AT2 receptor and guanylyl cyclase-A DKO (AT2/GCA-KO) mice were generated by Dr.
Nakao's group.18 Using this model, they showed that GCA inhibits AT2 receptor-mediated pro-hypertrophic signaling in
the heart.

VASCULAR DISEASE
We reported the potential beneficial effects of AT2 receptor signaling on vascular remodeling in a cuff placement
model,19,20 a wire injury model,21 and vascular senescence, in experiments using Agtr2− mice.22 Mice with smooth mus-
cle cell (VSMC)-specific overexpression of the AT2 receptor (smAT2-Tg) showed that the production of bradykinin is
stimulated and the nitric oxide (NO)/cGMP system is enhanced in a paracrine manner to promote vasodilation.23 On the
other hand, we also generated AT2 receptor and apolipoprotein E (ApoE)-DKO (AT2/ApoE-DKO) mice.24 Because the
AT2 receptor is located on the X chromosome, ApoE-KO mice and Agtr2− mice were bred to yield mice ­heterozygous
 Animal Models with a Genetic Alteration of AT2 Expression Chapter | 3  19

at the ApoE locus and heterozygous (female) or hemizygous (male) at the AT2 receptor locus. Using these DKO mice,
it was clearly proved that AT2 receptor stimulation plays an important role in atherosclerotic lesion formation through
regulation of oxidative stress,24 adipocyte differentiation, and adipose tissue dysfunction.25 Moreover, Takata et al.
generated smAT2-Tg and ApoE-knockout (smAT2-Tg/ApoE-KO) mice and demonstrated that vascular AT2 receptor
stimulation exerts antiatherogenic actions in an endothelial kinin/NO-dependent manner. Interestingly, such an anti-
oxidative effect is likely to be exerted by inhibition of the accumulation of superoxide-producing mononuclear leuko-
cytes.26 Moreover, Isbell et al. compared post-MI left ventricular remodeling among mice to overexpression of the AT2
receptor (AT2-Tg), deletion of the bradykinin subtype 2 receptor (B2R) (B2-KO), AT2-Tg with B2R deletion (AT2-Tg/
B2R-KO), and wild-type mice and found that attenuation of post-MI remodeling by overexpression of the AT2 receptor
is not directly mediated via the B2R pathway.27 Furthermore, double-gene-alteration mice generated by crossing Agtr2−
mice and overexpression of adipose tissue angiotensinogen (aAGT-Tg) were reported by Yvan-Charvet et al. These
mice exhibited higher blood pressure than that in aAGT-Tg mice. Interestingly, despite a reduction of adipose mass in
double-gene-alteration mice, AT2 receptor deficiency caused increased renin production.28 On the other hand, Agtr2−
mice bred with an established model of the Marfan syndrome, which are heterozygous for a cysteine substitution in
an epidermal growth factor-like domain of fibrillin-1 (Fbn1C1039G/+), exhibit exacerbated aortic diseases compared with
that in Marfan syndrome mice.29

RENAL DISEASE
Agtr2− mice exhibited a reduction in urinary sodium excretion via bradykinin and NO, against the antinatriuretic action
after infusion of Ang II.30 Obst et al. focused on renal NO synthase (NOS) isoforms. Agtr2− mice showed upregula-
tion of neuronal NOS (nNOS) and inducible NOS (iNOS).31 They also investigated the effect of deoxycorticosterone
acetate (DOCA)-salt on renal function using Agtr2− mice. Under DOCA-salt, renal iNOS was further increased and
could contribute to renal damage. Agtr2− mice showed increased renal fibrosis and fibrocyte infiltration with concomi-
tant upregulation of renal transcripts of procollagen type Iα1 (COL1A1) compared with wild-type mice, together with
an increased number of fibrocytes in the bone marrow.32 In the urinary tract, some Agtr2− mice show anomalies that
mimic human congenital anomalies of the kidney and urinary tract as a result of a defect of in utero organogenesis of
the excretory system.33 Moreover, the receptor for advanced glycation end products (RAGE) expression in podocytes
was moderately higher in Agtr2− mice than in control animals after Ang II treatment, indicating that Ang II-mediated
RAGE induction in podocytes occurs via the AT2 receptor.34 In contrast, Hashimoto et al. demonstrated protective roles
of the AT2 receptor against glomerular injury under the control of the α-SMA promoter, using AT2-Tg mice subjected
to 5/6 nephrectomy.35

NEURAL DISEASE
In brain development, von Bohlen und Halbach et al. reported an increase of cell number in the piriform cortex and hip-
pocampus CA1, CA2, and CA3 regions of Agtr2− mice compared with wild-type mice.36 By whole-genome microarray
analysis of RNA isolated from the brain of Agtr2− mice, it was shown that differentially expressed genes encode molecules
involved in multiple cellular processes including microtubule functions associated with dendritic spine morphology, sug-
gesting that AT2 receptor-modulated genes in the brain influence learning and memory.37 Agtr2− mice show anxiety-like
behavior involving the noradrenergic system. Okuyama et al. speculated that the amygdala appears to play an important
role in responses to fear and anxiety.38 Moreover, Watanabe et al. reported that Agtr2− mice exhibit a lower body tempera-
ture than controls, possibly due to a decrease in immunological stress-induced hyperthermia.39 On the other hand, the pain
threshold was significantly lower in Agtr2− mice, with a decreased level of brain beta-endorphins.40 These results indicate
that the AT2 receptor has various roles in behavior and brain functions.

CONCLUSION
Animal models with a genetic alteration of AT2 receptor expression have been discussed and are summarized in Table 1.
Genetic alteration animals contribute to functional analysis of not only the AT2 receptor but also interactions between other
receptors and renin–angiotensin system components.
 20  The Protective Arm of the Renin–Angiotensin System (RAS)
TABLE 1  Mouse Models with a Genetic Alteration of AT2 Receptor Expression

AT2 Receptor Conventional or

Modification Conditional Target Tissues Genetic Alteration Other Gene Alteration References

Deletion Conventional Whole body AT2R only [1,5–10,19–22,32,34]

Deletion Conventional Whole body AT2R only [2,3,11,30,31,33,36–40]

Overexpression Conditional Cardiac myocytes AT2R only [12,13]

Overexpression Conditional Smooth muscle cells AT2R only [23,35]

Overexpression Conditional Ventricular myocytes AT2R only [14–16]

Deletion Conventional Whole body Double Deletion of AT1a receptor (conventional) [5]

Deletion Conventional Whole body Double Deletion of ApoE (conventional) [24,25]

Overexpression Conditional Smooth muscle cells Double Deletion of ApoE (conventional) [26]

Overexpression Conventional Whole body Double Deletion of B2 receptor (conventional) [27]

Overexpression Conditional Cardiac myocytes Double Deletion of AT1a receptor (conventional) [17]

Deletion Conventional Whole body Double Overexpression of angiotensinogen (conditional; [28]

adipose tissue)

Deletion Conventional Whole body Double Deletion of guanylyl cyclase-A [18]

Deletion Conventional Whole body Double Heterozygous for a cysteine substitution in an [29]
epidermal growth factor-like domain of fibrillin-1
(conventional)

Deletion Conventional Whole body Triple Deletion of AT1a and AT1b receptors (conventional) [4]
 Animal Models with a Genetic Alteration of AT2 Expression Chapter | 3  21

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