Resistance to Neointimal Hyperplasia and Fatty StreakFormation in Mice With Adrenomedullin Overexpression
Yasushi Imai, Takayuki Shindo, Koji Maemura, Masataka Sata, Yuichiro Saito, Yukiko Kurihara,
Masahiro Akishita, Junichi Osuga, Shun Ishibashi, Kazuyuki Tobe, Hiroyuki Morita, Yoshio Oh-hashi,
Toru Suzuki, Hiromitsu Maekawa, Kenji Kangawa, Naoto Minamino, Yoshio Yazaki,
Ryozo Nagai, Hiroki Kurihara
Objective—Several in vitro studies have implicated that adrenomedullin (AM) plays an important role in the pathogenesisof vascular injury and fatty streak formation. To test this possibility in vivo, we evaluated 2 experimental models usingtransgenic mice overexpressing AM in a vessel-selective manner (AMTg mice).
Methods and Results—Placement of a periarterial cuff on femoral arteries resulted in neointimal formation at 2 to 4 weeks
to a lesser extent in AMTg mice than in their wild-type littermates (at 28 days, intima/media area ratio 0.45⫾0.14 versus
1.31⫾0.41, respectively; P⬍0.001). This vasculoprotective effect observed in AMTg mice was inhibited byN␻-nitro-L-arginine methyl ester. We further examined the effect of AM on hypercholesterolemia-induced fatty streak
formation by crossing AMTg mice with apolipoprotein E knockout mice (ApoEKO mice). The extent of the formationof fatty streak lesions was significantly less in ApoEKO/AMTg mice than in ApoEKO mice (percent lesion area
12.0⫾3.9% versus 15.8⫾2.8%, respectively; P⬍0.05). Moreover, endothelium-dependent vasodilatation as indicativeof NO production was superior in AMTg/ApoEKO mice compared with ApoEKO mice.
Conclusions—Taken together, our data demonstrated that AM possesses a vasculoprotective effect in vivo, which is atleast partially mediated by NO. (Arterioscler Thromb Vasc Biol. 2002;22:1310-1315.)
Key Words: adrenomedullin 䡲 transgenic mice 䡲 neointimal hyperplasia 䡲 fatty streak
䡲 apolipoprotein E knockout miceM

sin II and endothelin-1. However, direct evidence for such invivo roles has still not been well documented.
Recently, we established transgenic mice overexpressing
AM (AMTg mice) in a vessel-selective manner driven by themurine preproendothelin-1 promoter.10 AMTg mice exhibitedan increase in AM levels by 2- to 4-fold in the plasma andmajor organs and by 2- to 8-fold in the aorta by radioimmunoassay. Immunohistochemistry detected intense AM signals
in both ECs and VSMCs in the aorta and major arteries aswell as in arterioles in vascular-rich organs, including theheart, kidney, and lung. Concerning hemodynamics, chronic
AM overexpression in the vessel wall resulted in decreased
blood pressure through, at least in part, the stimulation of NO
production. We postulated that these mice could also be
useful for analyzing the role of AM in the pathophysiologicalprocess associated with vascular injury and atherosclerosis. In
the present study, we compared the formation of injuryinduced neointimal hyperplasia and hypercholesterolemia-

any vasoactive factors derived from endothelial cells(ECs) and vascular smooth muscle cells (VSMCs) are
known to regulate regional vascular tone as well as cellular
proliferation and migration, thereby affecting vascular structure. Dysregulated production of these factors is assumed tomake a contribution to pathological states, such as hypertension, atherosclerosis, and postangioplasty restenosis.
Adrenomedullin (AM), originally identified as a potent
vasodilatory peptide produced by human pheochromocytoma,1 has been shown to be released from ECs2 and, to a lesser
extent, VSMCs.3 In addition to its direct4 and NO-mediated5
vasodilatory activities, in vitro studies have demonstrated its
direct inhibitory effect on the migration6 and proliferation7 of
VSMCs and its antiapoptotic effect on ECs8,9 via NO.
Therefore, it is suggested that AM has a compensatory effecton deteriorating physiological status and a protective effectagainst vascular injury as a possible counterpart against
vasoconstrictive and mitogenic peptides, including angioten-

Received September 7, 2001; revision accepted May 20, 2002.
From the Department of Cardiovascular Medicine (Y.I., T. Shindo, K.M., M.S., Y.K., H. Morita, Y.O., T. Suzuki, H. Maekawa, R.N.) and theDepartment of Metabolic Diseases (J.O., S.I., K.T.), Graduate School of Medicine, The University of Tokyo, Tokyo; the 2nd Department of Internal
Medicine (Y.S.), School of Medicine, Gunma University, Maebashi; the Department of Geriatric Medicine (M.A.), School of Medicine, Kyorin
University, Tokyo; The National Cardiovascular Center Research Institute (K.K., N.M.), Suita; the International Medical Center of Japan (Y.Y.), Tokyo;
and the Division of Integrative Cell Biology (H.K.), Department of Embryogenesis, Institute of Molecular Embryology and Genetics, Kumamoto
University, Kumamoto, Japan.
Correspondence to Hiroki Kurihara, MD, PhD, Division of Integrative Cell Biology, Department of Embryogenesis, Institute of Molecular Embryology
and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan. E-mail kurihara@kaiju.medic.kumamoto-u.ac.jp© 2002 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org

1310

DOI: 10.1161/01.ATV.0000024685.92243.E7

Imai et al
induced fatty streak formation between mice with and without AM overproduction to clarify whether AM could be
involved in the protective mechanisms in the vasculature.

Methods
The experiments were performed in accordance with the Declarationof Helsinki and the University of Tokyo Institutional Guidelines for
Animal Experiments. The mice were housed in an animal room witha 12-hour light-dark cycle and a temperature of 22°C. The mice were
given standard chow and water ad libitum, unless otherwise
indicated.

Animals
The AMTg mouse line has already been established as previously
described. 10 We crossed AMTg mice with apoE knockout(ApoEKO) mice.11 To obtain AMTg/ApoEKO mice, homozygous
AMTg mice were bred with homozygous ApoEKO mice. The
resultant offspring, which overexpressed AM and were obligatorily
heterozygous for the mutant apoE locus, were further bred with eachother to obtain homozygous ApoEKO mice with or without the AM
transgene. The parental ApoEKO mice were F2 hybrids between
129/Sv and C57BL/6, and the parental AM transgenic mice originated from hybrids from the B6C3F1 and C3H strains, which were
backcrossed for at least 5 generations into the original background of
the ApoEKO mice.

Hemodynamics
We evaluated blood pressure with a programmable sphygmomanometer (BP98 Softron) by use of the tail-cuff method.12 All measurements were repeated 10 times for each animal, and the average wasobtained. To evaluate the effect of blood pressure reduction, several
mice were treated with hydralazine at a dose of 5 mg/kg per day intheir drinking water.13

Vascular Injury by Cuff Placement
We used 10- to 12-week-old AMTg and wild-type mice from thesame genetic background. The cuff placement surgery was performed according to a method described previously.14,15 In brief, themice were anesthetized with ketamine (70 mg/kg) and xylazine (4mg/kg) by intraperitoneal injection. The left femoral artery was
isolated from the surrounding tissues. A polyethylene tube (PE-50,
2 mm long, inner diameter 0.56 mm, outer diameter 0.965 mm;
Becton-Dickinson) was cut longitudinally, loosely placed around theartery, and closed with a suture. After the experimental period, themice were killed, and arterial tissues were fixed in 10% formalin andembedded in paraffin. The middle segment of the artery was cut into
5 subserial cross sections at intervals of 200 ␮m. The sections were
stained by elastica van Gieson or hematoxylin and eosin staining.
The areas of the neointima, media, and adventitia were measured byusing image-analyzing software (NIH image). The average of 5
sections was taken as a representative value for each animal. To
evaluate DNA synthesis, bromodeoxyuridine (BrdU, Sigma Chemical Co) was injected at doses of 100 mg/kg SC and 30 mg/kg IP 18
hours before euthanasia and then at a dose of 30 mg/kg IP 12 hours
before euthanasia.16 Immunohistochemistry using anti-BrdU antibody in serial sections was performed (BrdU Staining Kit, Zymed
Laboratories), and the BrdU index (the ratio of BrdU-positive nuclei
versus total nuclei) was calculated.
To evaluate the chronic inhibition of NO synthase, N␻-nitro-Larginine methyl ester (L-NAME) was dissolved in the drinking waterat a concentration of 200 mg/L17 for 28 days. Moreover, several mice
were treated with the AM receptor antagonist CGRP(8-37) toevaluate the effect of AM antagonism. Alzet micro-osmotic pumps(model 1002, Alza) were implanted intraperitoneally at the time of
cuff placement. The pumps delivered vehicle (saline) or CGRP(8-37)(20 mg/kg per day, Peptide Institute)18 continuously for 28 days at a
rate of 0.125 ␮L/h.

Vasculoprotective Effect of Adrenomedullin

1311

Evaluation of Fatty Streak Formation
Two diets were used: (1) a normal chow diet (MF diet from Oriental
Yeast Co) that contained 5.6% (wt/wt) fat with 0.09% (wt/wt)
cholesterol, and (2) an atherogenic diet, which consisted of the MF
diet containing 0.15% (wt/wt) cholesterol and 15% (wt/wt) butter.19
Eight-week-old ApoEKO/AMTg and ApoEKO mice were fed theatherogenic diet for 2 months and euthanized by an overdose ofanesthetic, and the extent of fatty streak formation was evaluated by
2 methods: (1) the en face surface lesion area20 and (2) thecross-sectional lesion area of aortic origin.21 In brief, for evaluationof the entire aorta, the aorta from the aortic sinus to the iliac
bifurcation was dissected, and lipid-rich atheroma were visualized bystaining with Sudan IV. As cross-sectional lesions of the aortic
sinuses, 4 serial sections at intervals of 60 ␮m were prepared, stainedwith oil red O, and counterstained with hematoxylin.

Vascular Reactivity to Endothelium-Dependentand -Independent Vasodilators
Vascular reactivity to endothelium-dependent and -independent vasodilators was tested by using the descending thoracic aorta toevaluate endothelial function as previously described.22 The aortic
rings were suspended under 1.0 g of tension and preconstricted withphenylephrine (5⫻10⫺8 mol/L). Acetylcholine (ACh) (10⫺9 to 10⫺5mol/L) and sodium nitroprusside (SNP, 10⫺8 to 10⫺5 mol/L) wereadded cumulatively to the organ bath. For ACh-induced vasodilatation after blockade of NO production, L-NAME (10⫺5 mol/L) was
administered to evaluate endogenous NO production. Data areexpressed as the percentage relaxation of phenylephrine-induced
preconstriction.

Statistical Analysis
Quantitative values are expressed as the mean⫾SD. Comparisons ofmeans were made by using the Student t test for unpaired values;
when ⬎2 means were compared, an ANOVA with repeated measurements was used. If a significant F value was found, the Scheffé
post hoc test for multiple comparisons was used to identify any
differences among groups. A value of P⬍0.05 was consideredsignificant, unless otherwise indicated. In aortic ring experiments,
the negative logarithm of the concentration of ACh or SNP that
produced half the putative maximal response was referred to as ED50.
Maximum vasorelaxation was shown as Emax (a percentage).

Results
Neointimal Formation Induced by Cuff Placement
We compared neointimal formation induced by cuff placement between AMTg and wild-type mice. In contralateral
sham-operated arteries, there was no neointima in either thewild-type or AMTg mice (Figure 1A). On the other hand, cuff
placement resulted in neointimal hyperplasia, which grew for
up to 28 days. Intimal hyperplasia was significantly smaller in
AMTg mice than in wild-type mice at 14 days and also at 28
days after surgery (Figure 1A). Twenty-eight days after
surgery, the ratio of the neointima area to the intimal/medial
area was 16 390⫾1890 ␮m2/1.31⫾0.41 in wild-type mice(n⫽10) and 5830⫾1620 ␮m2/0.45⫾0.14 in AMTg mice(n⫽10, P⬍0.001; Figure 1B).
For DNA synthesis of VSMCs, we examined BrdU uptake
3 and 7 days after injury. BrdU uptake in subendothelial
layers mainly composed of VSMCs was significantly higher
in the wild-type mice (n⫽7) than in the AMTg mice (n⫽7).
BrdU incorporation was relatively high within 1 week after
injury and almost completely diminished ⱖ14 days after
injury in AMTg and wild-type mice. These data indicate that
AM overexpression inhibited the proliferation of VSMCs(Figure 2A).

1312

Arterioscler Thromb Vasc Biol.

August 2002

Figure 2. A, BrdU uptake in the media and neointima of thecuffed femoral arteries. The BrdU index (the number of BrdU
positive nuclei/number of total nuclei) was analyzed 3 and 7
days after cuff placement. B, Number of inflammatory cells
invading around injured arteries. The average of 5 sections was
taken as the value for each animal. Error bars indicate SD.
#P⬍0.05. NS indicates not significant.

Figure 1. A, Photomicrographs showing representative crosssections of the femoral arteries 14 and 28 days after cuff placement. Sections of 5-␮m thickness were stained by elastica van
Gieson staining. B, Morphometric analysis of the cuffed femoral
arteries. Cross-sectional areas of media and intima were evaluated at 14 days and 28 days after cuff placement, and the intima/media ratio was calculated from the area of intima divided
by the area of media. *P⬍0.001.

Inflammatory cell infiltration around the vessels was prominently observed, especially within 1 week after injury. The
number of inflammatory cells was not different between
AMTg and wild-type mice at 3 and 7 days after injury,
indicating that the inflammatory process at the adventitia may
not be affected by AM overexpression (Figure 2B).

Effect of L-NAME, Hydralazine, and CGRP(8-37)
on Neointimal Hyperplasia
The systolic blood pressure measured by the tail-cuff method
was significantly lower in the AMTg mice than in thewild-type mice (101.2⫾9.3 versus 115.3⫾13.2 mm Hg, respectively; P⬍0.05). When L-NAME was chronically administered, the systolic blood pressure was significantly elevated,
and the difference between the 2 groups diminished(136.5⫾7.3 versus 141.2⫾11.4 mm Hg for AMTg versus
wild-type mice, respectively; P⫽NS). For the vascular injury,
the vasculoprotective effect in AMTg mice was diminished,
and there was no difference in the neointimal response

between the AMTg and wild-type littermates (Figure 3A),
suggesting that the vasculoprotective action of AM is mediated via an NO-dependent mechanism. Next, to confirm thatthe vasculoprotective effect of AM is independent of its
hypotensive effect, we evaluated the neointimal responses
between wild-type mice with and without hydralazine administration. The systolic blood pressure of wild-type micetreated with hydralazine was 98.3⫾8.5 mm Hg, which was
almost equivalent to that of AMTg mice. We evaluated theneointimal formation 28 days after injury in the wild-typemice treated with hydralazine, finding that the intima/media
ratio was 1.25⫾0.50, which was not significantly different
from that of wild-type mice without hydralazine treatment(1.31⫾0.41, P⫽NS; Figure 3B). Therefore, the protective
effect of AM was independent of the pressure reduction.
Moreover, to test the effect of AM antagonism, we treatedwild-type mice and AMTg mice with CGRP(8-37) or vehicle.
This demonstrated that CGRP(8-37) infusion partially inhibited the beneficial effect of AM overexpression comparedwith vehicle administration and did not significantly affectneointimal formation in the wild-type mice (Figure 3C).

Fatty Streak Formation in ApoEKO and
AMTg/ApoEKO MiceNext, we examined the protective effect of AM on fatty
streak formation by breeding AMTg with ApoEKO mice.
In AMTg/ApoEKO mice, AM transgene expression was
detected in the aorta, heart, lung, and kidney, as evaluatedby reverse transcription–polymerase chain reaction andNorthern blot analysis (data not shown), and plasma AM
levels evaluated by radioimmunoassay10 were significantly
higher in AMTg/ApoEKO mice than in ApoEKO miceImai et al

Figure 3. Effects of several drug interventions on neointimal formation. A, L-NAME. B, Hydralazine. C, CGRP(8-37). #P⬍0.05
and §P⬍0.01.

(8.36⫾2.32 versus 4.93⫾1.07 fmol/mL, respectively;
P⬍0.01; n⫽7 each).
ApoEKO mice and ApoEKO/AMTg mice were fed normalchow until they were 8 weeks old, and then they were fed theatherogenic diet for 2 months. Plasma lipid and lipoprotein
concentrations were markedly elevated after an atherogenic
diet. However, there were no significant differences in lipid
profiles between the AMTg/ApoEKO mice and ApoEKO
mice (data not shown). The AMTg/ApoEKO mice (n⫽16)
were significantly more hypotensive than the ApoEKO mice(n⫽16); systolic blood pressure was 110⫾15 versus
125⫾14 mm Hg, respectively (P⬍0.01). Thus, to evaluate
the direct effect of hypotension on fatty streak formation, we
also evaluated the hydralazine-treated ApoEKO mice, whose
systolic blood pressure (104⫾17 mm Hg) was almost equivalent to that of the AMTg/ApoEKO mice. The representative
photographs of en face surface atherosclerotic lesions areshown in Figure 4A. The fatty streak formation of the
AMTg/ApoEKO mice (n⫽12) was significantly smaller thanthat of the ApoEKO mice (n⫽12); the en face lesion area was
12.0⫾3.9% versus 15.8⫾2.8%, respectively (P⬍0.05, Figure
4B). The same result was observed in the cross-sectional
lesion area (223 000⫾56 000 versus 290 000⫾45 200 ␮m2
for AMTg/ApoEKO mice versus ApoEKO mice, respec-

Vasculoprotective Effect of Adrenomedullin

1313

Figure 4. Hypercholesterolemia-induced atherosclerotic lesions.
A, Representative photographs of en face atherosclerotic
lesions in the entire aorta. B, Percentage of en face atherosclerotic lesions. #P⬍0.05.

tively; P⬍0.01). However, the atheromatous lesion formation
was not changed in ApoEKO mice when they were treatedwith hydralazine (en face lesion area 16.2⫾3.1%, crosssectional lesion area 301 400⫾52 100 ␮m2; P⫽NS), suggesting that AM inhibits fatty streak formation via a mechanism
other than its hypotensive effect.

Endothelial Function and NO Production
To test endothelial function in ApoEKO/AMTg mice and
ApoEKO mice, we examined endothelium-dependent and-independent vasodilatation with the use of aortic rings
isolated from mice fed the atherogenic diet (n⫽8 for eachgroup, Figure 5). As controls, wild-type mice and AMTg
mice were used for the aortic ring experiment. There was no
significant difference in the tension after preconstriction byphenylephrine among the 4 groups. The relaxation induced by
ACh was significantly deteriorated in the apoE-deficient micecompared with the AMTg and wild-type mice. However,endothelium-dependent vasorelaxation was significantly better in ApoEKO/AMTg mice (Emax 94.6⫾2.5%, ED50 [⫺log
molar] 7.31⫾0.14) than in ApoEKO (Emax 85.2⫾4.6%, ED50
6.83⫾0.25; P⬍0.05). Concerning the 2 control groups, the1314

Arterioscler Thromb Vasc Biol.

August 2002

Figure 5. Endothelium-dependent vasorelaxation of aortas fromthe 4 groups: wild-type (closed triangle), AMTg (open square),
ApoEKO (closed square), and AMTg/ApoEKO (open circle) mice(n⫽8 to 10). Values are mean⫾SEM. #P⬍0.05.

ED50 was also significantly better in AMTg mice (7.39⫾0.30)
than in wild-type mice (7.77⫾0.16). However, there was no
significant difference in E max between AMTg mice
(97.7⫾2.4%) and wild-type mice (99.7⫾0.4%, P⫽NS). On
the other hand, there was no significant difference in aortic
relaxation in response to SNP among the 4 groups. AChinduced relaxation was almost completely attenuated by
L-NAME, suggesting that the vasorelaxation induced by ACh
is almost exclusively mediated by endothelium-derived NO.
These data suggest that endothelial function was preserved by
AM overexpression when damaged by remarkable hypercholesterolemia. Moreover, plasma cGMP levels were alsosignificantly higher in AMTg/ApoEKO mice (7.9⫾2.0 pmol/mL) than in ApoEKO mice (6.1⫾1.8 pmol/mL, P⬍0.05),
implicating that the steady-state NO production was also
upregulated in mice with AM overexpression. On the other
hand, the endothelial NO synthase (eNOS) expression levels
in aorta detected by reverse transcription–polymerase chain
reaction were not different between mice with and without
AM overexpression (data not shown), suggesting that thefunction of eNOS may be regulated by AM at the posttranslational or protein levels.

Discussion
In the present study, we examined the involvement of AM in
the formation of vascular lesions by analyzing 2 different
experimental models. We evaluated neointimal formationinduced by cuff placement and hypercholesterolemia-induced
fatty streak formation and found that mice carrying AMtransgenes were significantly resistant to vascular injury andfatty streak formation. This is the novel important evidence
that supports the vasculoprotective effect of AM in vivo.
Rodents are naturally resistant to neointimal hyperplasia
and fatty streak formation. Therefore, several experimental
models of neointimal hyperplasia or atherosclerosis havebeen proposed. In the present study, we adopted the cuffinjury model, in which the endothelial layer remains intact
and thrombus formation is very rare because no intravascular
manipulation is performed. Using this model, we demonstrated that AM overexpression suppressed neointimal hyper-

plasia and that BrdU-positive nuclei in the subendothelial
area were reduced in the cuff-injured femoral arteries, indicating that the proliferation of VSMCs was downregulated by
AM. This evidence is consistent with previous in vitro
experiments.7 On the other hand, there was no significant
difference in inflammatory cells around the injured arteries
between AMTg and wild-type mice, suggesting that inflammation may not be affected by AM overproduction.
AM can act on endothelial cells to stimulate NO production via
calcium-dependent activation of eNOS.5,23 We tested whether
inhibition of NO production diminished the protective effect of AM.
Chronic administration of L-NAME abolished the beneficial effectof AM overexpression on vascular injury. Therefore, the vasculoprotective effect of AM was thought to be at least partially mediatedby NO. The beneficial effect of NO produced by endothelium has
been previously demonstrated in many reports. eNOS knockout
mice have been reported to exhibit abnormal intimal hyperplasia
after vascular injuries in the cuff placement.14 eNOS overexpression
has also been shown to inhibit neointimal formation significantly inthe rat carotid balloon injury model.24 These previous studies andthe present result lead us to postulate that the activation of eNOS by
AM may be effective in preventing the formation of vascularproliferative lesions.
Shimizu et al18 reported that the AM antagonist CGRP(8-37)
inhibits neointimal hyperplasia after balloon injury in the rat
carotid artery, indicating that endogenous AM in the injured
tissue may promote the proliferation of VSMCs. This observation seems to contradict our data. In the present study, AM
overexpression inhibited neointimal formation, and CGRP(8-37)
infusion partially inhibited the beneficial effect of AM overexpression. This discrepancy may be due to differences in theexperimental animals and systems. In our 2 models, the endothelial layers remained intact, and AM was overexpressed
mainly in the endothelium. On the other hand, in a rat carotid
artery balloon-injury model, the endothelial layers were denuded, and the beneficial effect of AM on endothelial layers wasabolished. The injured VSMCs were exposed directly to theblood stream; thus, the phenotypic changes in VSMCs mightoccur more easily. Concerning other type of cells, AM has an
antiproliferative effect of glomerular mesangial cells25 but stimulates cell proliferation in Swiss 3T3 cells.26 Therefore, it islikely that the effect of AM on cell proliferation depends on thephenotypes or experimental situations.
To date, apoE knockout mice have been useful in the analysisof cholesterol-induced fatty streak formation. Several experiments on crossbreeding with other genetically altered mice or on
pharmacological intervention in ApoEKO mice have been reported.19,27 Thus, we crossbred AMTg mice with ApoEKO mice
to clarify the effect of AM on hypercholesterolemia-induced
fatty streak formation. We demonstrated that the presence ofAM transgenes suppresses the formation of atheromatous lesions and that this is independent of the blood pressure reduction
and lipoprotein profiles. It is worth noting that the vasculoprotective effect of AM was also confirmed in this model.
Endothelial dysfunction is characterized by impaired endothelium-derived NO-mediated vasorelaxation in atherosclerosis in humans and experimental animals. A previous
report28 demonstrated that endothelial dysfunction was impaired in isolated aortic rings of ApoEKO mice comparedImai et alwith wild-type mice, which was replicated in the present
study. In the present study, we found that endotheliumdependent vasodilatation was superior in AMTg/ApoEKO
mice compared with ApoEKO mice, although severe dyslipidemia damaged endothelium-dependent vasorelaxation significantly. Moreover, the plasma cGMP level (taken to be
indicative of steady-state NO production) was significantly
higher in ApoEKO/AMTg mice than in ApoEKO mice.
Therefore, our data suggest that the antiatherogenic effect of
AM in ApoEKO is at least partially caused by endotheliumderived NO production by chronic AM overexpression.
In summary, we demonstrated the beneficial effects of AM
in vivo against vascular injury at least partially via endothelial NO production with the use of genetically altered mice.
However, further studies are needed to clarify the precise
mechanism of the vasculoprotective effect of AM so that AM
can be applied clinically for atherosclerosis and for postangioplasty neointimal hyperplasia.

Acknowledgments
T.S. and Y.K. are Research Fellows in the Program for Promotion of
Fundamental Studies in Health Sciences of the Organization for
Drug ADR Relief, R&D Promotion, and Product Review of Japan.
This work was supported by the Japan Society for the Promotion of
Science Research for the Future Program; grants-in-aid for scientificresearch from the Ministry of Education, Science, and Culture,
Japan; a research grant for cardiovascular diseases (11C-1) from the
Ministry of Health and Welfare; and the Program for Promotion of
Fundamental Studies in Health Sciences of the Organization for
Drug ADR Relief, R&D Promotion, and Product Review of Japan.
We thank Stephen McKay for his critical reading of the manuscript.

References
1. Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, Eto
T. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun. 1993;192:553–560.
2. Sugo S, Minamino N, Kangawa K, Miyamoto K, Kitamura K, Sakata J,
Eto T, Matsuo H. Endothelial cells actively synthesize and secrete
adrenomedullin. Biochem Biophys Res Commun. 1994;201:1160 –1166.
3. Sugo S, Minamino N, Shoji H, Kangawa K, Kitamura K, Eto T, Matsuo
H. Production and secretion of adrenomedullin from vascular smoothmuscle cells: augmented production by tumor necrosis factor-alpha.
Biochem Biophys Res Commun. 1994;203:719 –727.
4. Nuki C, Kawasaki H, Kitamura K, Takenaga M, Kangawa K, Eto T,
Wada A. Vasodilator effect of adrenomedullin and calcitonin gene-related
peptide receptors in rat mesenteric vascular beds. Biochem Biophys Res
Commun. 1993;196:245–251.
5. Hirata Y, Hayakawa H, Suzuki Y, Suzuki E, Ikenouchi H, Kohmoto O,
Kimura K, Kitamura K, Eto T, Kangawa K. Mechanisms ofadrenomedullin-induced vasodilation in the rat kidney. Hypertension.
1995;25:790 –795.
6. Kohno M, Yokokawa K, Kano H, Yasunari K, Minami M, Hanehira T,
Yoshikawa J. Adrenomedullin is a potent inhibitor of angiotensin
II-induced migration of human coronary artery smooth muscle cells.
Hypertension. 1997;29:1309 –1313.
7. Kano H, Kohno M, Yasunari K, Yokokawa K, Horio T, Ikeda M, Minami
M, Hanehira T, Takeda T, Yoshikawa J. Adrenomedullin as a novel
antiproliferative factor of vascular smooth muscle cells. J Hypertens.
1996;14:209 –213.
8. Kato H, Shichiri M, Marumo F, Hirata Y. Adrenomedullin as an autocrine/paracrine apoptosis survival factor for rat endothelial cells. Endocrinology. 1997;138:2615–2620.
9. Sata M, Kakoki M, Nagata D, Nishimatsu H, Suzuki E, Aoyagi T,
Sugiura S, Kojima H, Nagano T, Kangawa K, Matsuo H, Omata M, Nagai
R, Hirata Y. Adrenomedullin and nitric oxide inhibit human endothelial

Vasculoprotective Effect of Adrenomedullin

1315

cell apoptosis via a cyclic GMP-independent mechanism. Hypertension.
2000;36:83– 88.
10. Shindo T, Kurihara H, Maemura K, Kurihara Y, Kuwaki T, Izumida T,
Minamino N, Ju KH, Morita H, Oh-hashi Y, Kumada M, Kangawa K,
Nagai R, Yazaki Y. Hypotension and resistance to lipopolysaccharideinduced shock in transgenic mice overexpressing adrenomedullin in theirvasculature. Circulation. 2000;101:2309 –2316.
11. Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E.
Science. 1992;258:468 – 471.
12. Takimoto E, Ishida J, Sugiyama F, Horiguchi H, Murakami K, Fukamizu
A. Hypertension induced in pregnant mice by placental renin andmaternal angiotensinogen. Science. 1996;274:995–998.
13. Hayek T, Attias J, Coleman R, Brodsky S, Smith J, Breslow JL, Keidar
S. The angiotensin-converting enzyme inhibitor, fosinopril, and the angiotensin II receptor antagonist, losartan, inhibit LDL oxidation andattenuate atherosclerosis independent of lowering blood pressure in apolipoprotein E deficient mice. Cardiovasc Res. 1999;44:579 –587.
14. Moroi M, Zhang L, Yasuda T, Virmani R, Gold HK, Fishman MC, Huang
PL. Interaction of genetic deficiency of endothelial nitric oxide, gender,
and pregnancy in vascular response to injury in mice. J Clin Invest.
1998;101:1225–1232.
15. Akishita M, Horiuchi M, Yamada H, Zhang L, Shirakami G, Tamura K,
Ouchi Y, Dzau VJ. Inflammation influences vascular remodeling throughAT2 receptor expression and signaling. Physiol Genomics. 2000;2:13–20.
16. Nakajima M, Hutchinson HG, Fujinaga M, Hayashida W, Morishita R,
Zhang L, Horiuchi M, Pratt RE, Dzau VJ. The angiotensin II type 2 (AT2)
receptor antagonizes the growth effects of the AT1 receptor: gain-offunction study using gene transfer. Proc Natl Acad Sci U S A. 1995;92:10663–10667.
17. Ribeiro MO, Antunes E, de Nucci G, Lovisolo SM, Zatz R. Chronic
inhibition of nitric oxide synthesis: a new model of arterial hypertension.
Hypertension. 1992;20:298 –303.
18. Shimizu K, Tanaka H, Sunamori M, Marumo F, Shichiri M.
Adrenomedullin receptor antagonism by calcitonin gene-related
peptide(8-37) inhibits carotid artery neointimal hyperplasia after balloon
injury. Circ Res. 1999;85:1199 –1205.
19. Yagyu H, Ishibashi S, Chen Z, Osuga J, Okazaki M, Perrey S, Kitamine T,
Shimada M, Ohashi K, Harada K, Shionoiri F, Yahagi N, Gotoda T, Yazaki Y,
Yamada N. Overexpressed lipoprotein lipase protects against atherosclerosis inapolipoprotein E knockout mice. J Lipid Res. 1999;40:1677–1685.
20. Tangirala RK, Rubin EM, Palinski W. Quantitation of atherosclerosis in murine
models: correlation between lesions in the aortic origin and in the entire aorta, anddifferences in the extent of lesions between sexes in LDL receptor-deficient andapolipoprotein E-deficient mice. J Lipid Res. 1995;36:2320–2328.
21. Paigen B, Morrow A, Holmes PA, Mitchell D, Williams RA. Quantitative
assessment of atherosclerotic lesions in mice. Atherosclerosis. 1987;68:231–240.
22. Saito Y, Yamagishi T, Nakamura T, Ohyama Y, Aizawa H, Suga T,
Matsumura Y, Masuda H, Kurabayashi M, Kuro-o M, Nabeshima Y,
Nagai R. Klotho protein protects against endothelial dysfunction.
Biochem Biophys Res Commun. 1998;248:324 –329.
23. Miura K, Ebara T, Okumura M, Matsuura T, Kim S, Yukimura T, Iwao H.
Attenuation of adrenomedullin-induced renal vasodilatation by NG-nitro
L-arginine but not glibenclamide. Br J Pharmacol. 1995;115:917–924.
24. von der Leyen HE, Gibbons GH, Morishita R, Lewis NP, Zhang L,
Nakajima M, Kaneda Y, Cooke JP, Dzau VJ. Gene therapy inhibiting
neointimal vascular lesion: in vivo transfer of endothelial cell nitric oxide
synthase gene. Proc Natl Acad Sci U S A. 1995;92:1137–1141.
25. Parameswaran N, Nambi P, Brooks DP, Spielman WS. Regulation ofglomerular mesangial cell proliferation in culture by adrenomedullin. Eur
J Pharmacol. 1999;372:85–95.
26. Withers DJ, Coppock HA, Seufferlein T, Smith DM, Bloom SR,
Rozengurt E. Adrenomedullin stimulates DNA synthesis and cell proliferation via elevation of cAMP in Swiss 3T3 cells. FEBS Lett. 1996;378:
83– 87.
27. Knowles JW, Reddick RL, Jennette JC, Shesely EG, Smithies O, Maeda
N. Enhanced atherosclerosis and kidney dysfunction in eNOS(-/-)
ApoE(-/-) mice are ameliorated by enalapril treatment. J Clin Invest.
2000;105:451– 458.
28. Wang YX, Halks-Miller M, Vergona R, Sullivan ME, Fitch R, Mallari C,
Martin-McNulty B, da Cunha V, Freay A, Rubanyi GM, Kauser K.
Increased aortic stiffness assessed by pulse wave velocity in apolipoprotein E-deficient mice. Am J Physiol. 2000;278:H428 –H434.