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AT2 Receptor and Vascular Smooth Muscle Cell
Differentiation in Vascular Development
Hiroyuki Yamada, Masahiro Akishita, Masaaki Ito, Kouichi Tamura, Laurent Daviet,
Jukka Y.A. Lehtonen, Victor J. Dzau, Masatsugu Horiuchi
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Abstract—The angiotensin II type 2 (AT2) receptor is transiently expressed at late gestation in the fetal vasculature, but its
expression rapidly declines after birth. We have previously demonstrated that the expression of this receptor mediates
decline in vascular DNA synthesis that occurs at this stage of vascular development. To examine further the role of the
AT2 receptor in vasculogenesis, we have focused on the effect of the AT2 receptor on vascular smooth muscle cell
(VSMC) differentiation. In this study, we examined the time-dependent expression of differentiation markers for
VSMCs in the aorta of wild-type and AT2 receptor–null mice. a-Smooth muscle actin was expressed at the early stage
of differentiation and exhibited unchanged expression before and after the peak of AT2 receptor expression, which was
observed at embryonic day 20, neonatal day 1, and thereafter. No difference in a-smooth muscle actin expression was
observed between the wild-type and AT2 receptor–null mice. In contrast, the mRNA levels for calponin, expressed in
the late stage of VSMC differentiation, were significantly higher in the wild-type mouse aorta as compared with the AT2
receptor–null mice, which correlates with expression of the AT2 receptor. Moreover, the protein levels of calponin and
high-molecular-weight caldesmon (h-caldesmon) showed lower expression in the aorta of AT2 receptor knockout mice
at 2 and 4 weeks after birth. Taken together, our results suggest that the AT2 receptor promotes vascular differentiation
and contributes to vasculogenesis. (Hypertension. 1999;33:1414-1419.)
Key Words: angiotensin n cell differentiation n receptors, angiotensin II n muscle, smooth, vascular
n human development
A
and remodeling in late gestation. Indeed, pharmacological
blockade of the vascular AT2 receptor by use of the
specific antagonist PD123319 in the rat fetus during rat
embryonic days 16 to 21 (E16-E21) has resulted in
decreased aortic DNA synthesis, which supports a contribution of the AT2 receptor to vascular development.
We and others have obtained, by use of a homologous
recombination, AT2 receptor knockout mouse strains that
exhibit enhanced pressor response to acute Ang II infusion
as compared with the wild-type strain.7,8 Because the
vascular AT2 receptor is minimally expressed at the time
the blood pressure and Ang II infusion studies were
performed, the data suggest that the transient and developmentally regulated AT2 receptor expression exerts a
long-term effect on blood pressure, possibly through its
influence on vascular structure and/or function. These
results support the notion that the AT2 receptor modulates
the growth of the developing blood vessel and thus
contributes to vascular remodeling in late gestation.
Little is known about the ability of the AT2 receptor to
modulate VSMC differentiation. Recent studies suggest
that AT2 receptor activation may enhance differentiation in
PC12W cells, a rat pheochromocytoma cell line,9 –11 and
ngiotensin II (Ang II), a key regulator of cardiovascular homeostasis, exerts various actions in its diverse target tissues, that controls vascular tone, hormone
secretion, tissue growth, and neuronal activity. Two major
isoforms of the Ang II receptor, type 1 (AT1) and type 2
(AT2), have been defined on the basis of their ligand
selectivity. Most of the known effects of Ang II in the adult
tissues are attributable to the AT1 receptor. The AT2
receptor is abundantly and widely expressed in fetal
tissues,1–3 and its expression declines rapidly after birth,
which suggests that the AT2 receptor is involved in growth,
development, and/or differentiation.
The expression of the AT2 receptor in rat fetal blood
vessel is “turned on” at late gestation (embryonic days 16
to 21) and in the early neonatal period but decreases
rapidly to very low levels in the adult vessel.4,5 We have
demonstrated that this receptor exerts growth-modulatory
effects in vascular smooth muscle cells (VSMCs) such as
the inhibition of DNA synthesis and the induction of
apoptosis.4,6 The spatial and temporal patterns of the rat
vascular AT2 receptor expression, together with the growth
modulatory action, have led us to hypothesize that this
receptor plays an important role in vascular development
Received September 21, 1998; first decision October 15, 1998; revision accepted February 2, 1999.
From Cardiovascular Research, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass.
Correspondence to Masatsugu Horiuchi, MD, PhD, Department of Medical Biochemistry, Ehime University School of Medicine, Sigenobu, Onsen-gun,
Ehime 791-0295, Japan. E-mail [email protected]
© 1999 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
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NG 108-15 cells.12 The middle-sized neurofilament subunit expression was reduced in PC12W cells by AT2
receptor stimulation.10 In quiescent PC12W cells, AT2
receptor stimulation upregulated microtubule components
and polymerized b-tubulin and MAP2, but downregulated
MAP1B protein levels and, in PC12W cells differentiated
by nerve growth factor, AT2 receptor stimulation elevated
polymerized b-tubulin and reduced MAP1B.11 The morphological changes of AT2 receptor–mediated neurite outgrowth in NG 108-15 cells were correlated with an
increase in the level of polymerized tubulin and in the level
of the microtubule-associated protein.11 These results suggest a specific role of AT2 receptors in neuronal cell
differentiation through regulation of the cytoskeleton.
Thus it is conceivable that the AT2 receptor exerts a similar
effect on smooth muscle cell differentiation. The AT2
receptor–null mouse model provides a unique opportunity
to address the physiological role of the AT2 receptor.
According to this hypothesis, activation of the AT2 receptor would facilitate VSMC differentiation in the wild-type
animal, whereas the disruption of the AT2 receptor in the
knockout animals will result in the delayed differentiation
of VSMCs.
Methods
Animals
Female mice heterozygous for the AT2 receptor mutant allele7 were
mated with WT FVB/N male mice (Jackson Laboratory, Bar Harbor,
Maine). Pregnant mice were killed with an overdose of anesthesia,
and the fetuses were dissected from uterine decidua. The day when
a vaginal plug was observed was considered E1. In addition,
postnatal and 2-week-old, 4-week-old, and 8-week-old male mice,
which were obtained by the same mating as the fetuses, were used.
These fetuses and postnatal mice, back-crossed for 6 generations into
the FVB/N background, had 98% FVB/N and 2% 129/SV background. Animal genotyping was performed as previously described,7
with the use of body or tail genomic DNA samples. The animals
were housed in a room in which light was controlled (12 hours on,
12 hours off) and room temperature was kept at 22°C. They were
given standard diet and water ad libitum. All experimental procedures were approved and carried out in accordance with the guideline
of the Harvard Medical Area Standing Committee on Animals.
Immunoblot Analysis
Mice were anesthetized with an intraperitoneal injection of ketamine
(70 mg/kg) and xylazine (4 mg/kg); the thorax was opened and the
aorta was removed. The connective tissues around the thoracic aorta
were carefully dissected away in ice-cold phosphate-buffered saline
(pH 7.40) under stereomicroscopy. Tissue samples were homogenized in 50 mmol/L Tris-HCl buffer, pH 7.5, which contained
150 mmol/L NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate,
0.1% SDS, 1 mmol/L PMSF, 1.5 mmol/L aprotinin, 5.5 mmol/L
leupeptin, and 5.5 mmol/L pepstatin A. Tissue lysates were centrifuged at 10 000g for 20 minutes, boiled in Laemli loading buffer for
3 minutes, resolved by 8% SDS-PAGE, electroblotted onto nitrocellulose membrane, and immunoblotted with antibodies against highmolecular-weight caldesmon (h-caldesmon) (clone C21),13 calponin
(clone hCP),14 or a-smooth muscle actin (a-SM actin) (clone 1A4)15
(Sigma Chemical Co). Antibodies were detected by horseradish
peroxidase–linked secondary antibody with the use of an enhanced
chemiluminescence system (Amersham Life Science Inc). Densitometric analysis was performed by scanning densitometer (GS300,
Hoeffer) and NIH image software.
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Reverse Transcription–Polymerase Chain Reaction
Pregnant mice were anesthetized with an intraperitoneal injection of
ketamine (70 mg/kg) and xylazine (4 mg/kg) at 13, 15, 18, and 20
days of gestation (E13, E15, E18, and E20). Fetuses were removed
and their thoracic aortas were carefully excised under stereomicroscopy, frozen in liquid nitrogen, and kept at 280°C before use. Aortas
were also prepared from mice at 1, 7, 14, and 28 days after birth.
Total RNA was prepared from 4 to 7 pooled thoracic aortas for each
group with the use of TRIzol reagent (GIBCO BRL). Total RNA was
first treated with RNAse-free DNAse (0.02 U/mL). After treatment
for 5 minutes at 94°C, the samples were subjected to reverse
transcription with random hexamer primers and reverse transcriptase
(GeneAmp RNA PCR Kit, Perkin Elmer). We performed other sets
of reverse transcription–polymerase chain reaction (RT-PCR) without RNA samples to confirm that there was no artificial amplification
caused by the contaminations in the reagents. PCR primers were as
follows: calponin, 59-CACCAACAAGTTTGCCAG-39 and 59TGTGTCGCAGTGTTCCAT-3914; a-SM actin, 59-GAGAAGCCCAGCCAGTCG-39 and 59-CTCTTGCTCTGGGCTTCA-3916; AT1
receptor, 59-GGAAACAGCTTGGTGGTG-39 and 59-CTGAATTTCATAAGCCTTCTT-3917; AT2 receptor, 59-AGTGCATGCGGGAGCTG-39 and 59-GACAACAAAACAGTGAG-3918; and GAPDH,
59-ATGGTGAAGGTCGGTGTG-39 and 59-ACCAGTGGATGCAGGGAT-39.19 PCR reactions for a-SM actin, calponin, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were performed with
25 cycles of 1 minute of denaturation at 94°C, 1 minute of annealing
at 53°C, and 2 minutes of extension at 72°C followed by 10 minutes
of final extension step. PCR reactions for AT1 or AT2 receptors were
carried out with 30 cycles. We observed a linear increase in
amplification of PCR products with an increased amount of RNA up
to 2 mg and as well as the increase in PCR cycles until 30 cycles for
a-SM actin, calponin, or GAPDH and until 35 cycles for AT1 and
AT2 receptors. PCR products were separated by 1% agarose gel
electrophoresis. To verify the identity of the PCR products, we
sequenced PCR products and confirmed that the sequences of PCR
products matched up to the predicted sequences.
Blood Pressure Measurements
Male mice were anesthetized and a PE10 tube (Becton Dickinson)
was inserted into the left common carotid artery. The next day, the
PE50 tube was connected to the carotid artery cannula. The other end
of the tube was connected to the blood pressure transducer (model
TRN050, Kent Scientific Corp), which was connected to a transducer
amplifier (model ETH-400, CB Sciences, Inc) and a MacLab 4/s
(Division of AD Instruments, Inc). Blood pressure and heart rate
were measured with rats in a conscious, unrestrained condition.
Data Analysis
All values are expressed as mean6SE. Statistical significance for the
developmental changes in differentiation marker expression in each
strain of mice was assessed by ANOVA followed by Bonferroni test.
Student’s t tests were used for statistical comparisons between 2
strains of mice. A value of P,0.05 was considered to be significant.
Results
To study the effect of AT2 receptor gene disruption on VSMC
differentiation in the aorta, we examined the VSMC-specific
contractile proteins as differentiation markers at different
ages before birth and up to 8 weeks after birth. As shown in
Figure 1, the protein contents of h-caldesmon and calponin
increase age dependently. These protein levels were significantly lower in AT2 receptor–null mice than those in wildtype mice at 2 and 4 weeks of age (Figure 1, A, B, and C).
Interestingly, at 8 weeks of age there were no differences in
these marker proteins between both strains. On the other
hand, there were no differences in a-SM actin expression
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Figure 1. Developmental changes in expression of h-caldesmon, calponin, and a-SM actin in thoracic aorta. Tissue lysates were prepared from thoracic aortas from AT2-null (Null) and wild-type (WT) mice at the ages of 2, 4, and 8 weeks, separated on SDS/PAGE,
electroblotted on the nitrocellulose membrane, and immunoblotted with h-caldesmon, calponin, or a-SM actin antibodies. A, Representative data obtained from 8 different experiments. B, C, and D, Signal density is represented as percentage of the value obtained in
wild-type mice at 8 weeks of age. Values are expressed as mean6SE obtained from 8 animals in each group. *P,0.05 vs wild-type
mice at the same age.
between the strains throughout development (Figure 1, A
and D).
Next, we examined the developmentally regulated calponin, a-SM actin, and AT1 and AT2 receptor mRNA
expressions in the fetal aorta by RT-PCR. To show the
linear increase of RT-PCR products with increased
amounts of RNA by use of our PCR conditions described
in Methods, total RNA (2, 1, or 0.5 mg of RNA) prepared
from thoracic aorta of wild-type mice at the age of E20 was
subjected to RT-PCR for AT1 receptor, AT2 receptor,
calponin, a-SM actin, and GAPDH (Figure 2). In the
following experiments shown in Figure 3, we applied 1 mg
RNA to RT-PCR. The mouse AT1 receptor mRNA is
expressed at relatively constant levels from the initial age
tested (E13) throughout development both in AT2 receptor–null and wild-type mice (Figure 3A). The AT2 receptor
mRNA expression was observed at E13, increased thereafter, and rapidly declined after birth (Figure 3A). As
shown in Figure 3B, the mRNA expression of calponin in
wild-type mice was observed at E13, associated with the
onset of AT2 receptor mRNA expression, and increased
thereafter. The expression and the developmental increase
in calponin mRNA expression in AT2 receptor–null mice
were significantly delayed. Aortic calponin mRNA expression in the wild-type and AT2 receptor–null mouse appeared to be similar at 14 and 28 days after birth (Figure
3C). In contrast, a-SM actin mRNA level did not differ
throughout development between the 2 strains.
Discussion
AT2 receptor is expressed in fetal aorta during late gestation;
its expression decreases rapidly after birth, and the AT2
receptor is reexpressed in vascular injury.4 We have demonstrated that pharmacological blockade of the AT2 receptor in
fetal rats E16 to E21 by use of the specific antagonist
PD123319 has resulted in decreased DNA synthesis of the
developing aorta.4 Accordingly, we have hypothesized that
the AT2 receptor is involved with vascular development. In
this study we examined the possibility that the AT2 receptor
modulates VSMC differentiation and plays a role in vasculogenesis. Our data demonstrated that the protein levels of
h-caldesmon and calponin in VSMC in thoracic aorta of AT2
receptor–null mice were significantly lower than those in
wild-type mice up to 4 weeks of age, whereas the expression
of a-SM actin did not show any difference between the 2
strains. Expressions of h-caldesmon and calponin as well as
a-SM actin are developmentally regulated in VSMC.20 –22
a-SM actin appears in the early stage in vasculogenesis,
whereas the expressions of h-caldesmon and calponin begin
later and have been used as markers of VSMC differentiation
in the late stage of vasculogenesis.
To examine the relation of AT2 receptor expression with
VSMC differentiation, we examined h-caldesmon, calponin, a-SM actin, and AT2 receptor expression in fetal and
neonatal mice. Consistent with previous data on the rat,4,5
we demonstrated that mouse AT2 receptor mRNA expression is low during early development (E13-E15) but
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Figure 2. RT-PCR showing semiquantitative evaluation of
mRNAs for AT1 receptor, AT2 receptor, calponin, a-SM actin,
and GAPDH. Total RNA was prepared from thoracic aorta of
wild-type mice at the age of E20; 2 mg (lane 1), 1 mg (lane 2),
or 0.5 mg (lane 3) of RNA was subjected to RT-PCR. M, 100-bp
ladder DNA marker.
increases significantly during later stage of development
(E18-E20) and in the neonate (1 day after birth), whereas
the AT1 receptor is constantly expressed through early
embryonic stage after birth. The mRNA expression of
calponin in wild-type mice aorta was “switched on” at E13
to E15 in both strains of mouse. The level of calponin
mRNA increased rapidly thereafter in wild-type mouse
aorta, whereas calponin mRNA in the AT2 receptor–null
mouse was significantly lower and reached similar levels
of calponin mRNA in the wild-type mouse at 14 days after
birth. These results suggest that the level of expression of
calponin is closely associated with the AT2 receptor
expression in the fetal aorta. Moreover, we demonstrated
that the protein levels of calponin and h-caldesmon are
significantly higher in the AT2 receptor–null mouse aorta
up to 4 weeks after birth. In contrast, we did not observe
any significant difference in a-SM actin mRNA expression
in aorta between the 2 strains during development. a-SM
actin expression was already observed at E13, when the
AT2 receptor was not expressed, which suggests that a-SM
actin is not regulated by the AT2 receptor. These findings
suggest strongly that the AT2 receptor plays a role in the
phenotypic differentiation of VSMC, especially in the late
stage of gestation.
We did not directly measure VSMC differentiation or
vasculogenesis. h-caldesmon and calponin, thin filament–
associated proteins, have been suggested to modulate
smooth muscle contractility.23–26 Binding of h-caldesmon
and calponin to actin inhibits actomyosin ATPase, which is
necessary for initiation of contraction of VSMC and
appears to be involved in vascular contraction in vivo.27–29
Moreover, h-caldesmon and calponin have been reported
to inhibit contraction by providing a basal resting inhibi-
Figure 3. Developmental changes in mRNA expression of (A)
AT1 and AT2 receptors, (B) calponin, a-SM actin, and AT2 receptor, and (C) calponin in mouse aorta. Total RNA (1 mg) was prepared from thoracic aortas of AT2 receptor–null (Null) and wildtype (WT) mice at the ages of E13, E15, E18, E20, N1, 1, 2, and
4 weeks and subjected to RT-PCR. Results show representative
data of 3 separate experiments. GAPDH was used as internal
control42, 43 to standardize the amount of total RNA used.
tion of vascular tone in smooth muscle cells.24,26 To
examine the possibility that delayed expression of
h-caldesmon and calponin in AT2 receptor–null mice
influences the resting vascular tone and blood pressure, we
measured blood pressure and heart rate in conscious mice
at 4 and 8 weeks of age. Basal mean blood pressure in
4-week-old AT2 receptor–null mice was significantly
higher than that in wild-type mice (105.264.5 versus
87.563.9 mm Hg, n510, P,0.05), whereas there was no
difference in blood pressure at 8 weeks of age (109.265.7
versus 107.764.8 mm Hg, n510). There were no significant differences in heart rate between both strains. Mean
blood pressure of AT2 receptor–null mice was significantly
higher compared with wild-type mice at the age of 4
weeks, when h-caldesmon and calponin contents are lower
in AT2 receptor–null mice. Along with the increase in
h-caldesmon and calponin in AT2 receptor–null mouse
aorta, the blood pressure difference disappeared at age 8
weeks. These results suggest the possibility that the
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delayed expression of these VSMC markers in AT2 receptor null–mice plays some role in vasculogenesis and
influence vascular contractility.
We have demonstrated that the AT2 receptor exerts
antigrowth and proapoptotic effects in VSMC in vitro and
in vivo.4,6,30 The antigrowth effect of the AT2 receptor in
endothelial cells and cardiomyocytes have also been reported.31,32 In this report, we postulate that the AT2
receptor enhances the differentiation of VSMC in aorta.
Indeed, it has been reported that the AT2 receptor promotes
differentiation of neuronal cells through regulation of
cytoskeletal proteins.9 –12 The AT2 receptor has been reported to stimulate protein tyrosine phosphatase (PTPase)
activity,4,6,33–37 which plays an essential role in hematopoietic cell differentiation.38 We have reported that the
intracellular third loop of the AT2 receptor is important for
its PTPase activation.30 Interestingly, recent studies suggest that dopamine receptors, which have a similar amino
acid motif in the intracellular third loop,39 promotes
morphogenesis of developing neurons.40,41 These findings
suggest a possible mechanism whereby AT2 receptor–
regulated PTPase is involved in the differentiation of
VSMC. Our results also suggest that AT2 receptor stimulation did not modify the expression of housekeeping
genes such as GAPDH.42,43 In summary, our results suggest
that the AT2 receptor participates in mouse vasculogenesis.
Acknowledgments
This work was supported by National Institutes
HL-46631, HL-35252, HL-35610, HL-48638,
HL-58616 and a grant from the Longwood
Translational Research. Dr Dzau is a recipient
Award HL-35610.
of Health grants
HL-07708, and
Foundation for
of NIH MERIT
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AT2 Receptor and Vascular Smooth Muscle Cell Differentiation in Vascular Development
Hiroyuki Yamada, Masahiro Akishita, Masaaki Ito, Kouichi Tamura, Laurent Daviet, Jukka Y.
A. Lehtonen, Victor J. Dzau and Masatsugu Horiuchi
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Hypertension. 1999;33:1414-1419
doi: 10.1161/01.HYP.33.6.1414
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