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Published online before print November 4, 2002, 10.1161/01.CIR.0000042707.50032.19
(Circulation. 2002;106:2767.)
© 2002 American Heart Association, Inc.
Brief Rapid Communications |
Yoshihisa Okamoto, MD; Shinji Kihara, MD, PhD;Noriyuki Ouchi,
MD, PhD; Makoto Nishida, MD, PhD; Yukio Arita, MD, PhD; Masahiro
Kumada, MD; Koji Ohashi, MD; Naohiko Sakai, MD, PhD; Iichiro
Shimomura, MD, PhD; Hideki Kobayashi, MS; Naoki Terasaka, MS;
Toshimori Inaba, MS; Tohru Funahashi, MD, PhD; Yuji Matsuzawa,
MD, PhD
From the Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan (Y.O., S.K., N.O., M.N., Y.A., M.K., K.O., N.S., I.S., T.F., Y.M.); and Sankyo Co, Ltd, Shinagawa-ku, Tokyo, Japan (H.K., N.T., T.I.).
Correspondence to Shinji Kihara, Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, 2-2 B5 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail kihara@imed2.med.osaka-u.ac.jp
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Abstract |
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Background— Dysregulation of adipocyte-derived
bioactive molecules plays an important role in the
development of atherosclerosis. We previously reported
that adiponectin, an adipocyte-specific plasma
protein, accumulated in the injured artery from the plasma and
suppressed endothelial inflammatory response and vascular smooth
muscle cell proliferation, as well as macrophage-to-foam cell
transformation in vitro. The current study investigated whether
the increased plasma adiponectin could actually reduce atherosclerosis
in vivo.
Methods and Results— Apolipoprotein
E-deficient mice were treated with recombinant
adenovirus expressing human adiponectin (Ad-APN) or
ß-galactosidase (Ad-ßgal). The plasma adiponectin
levels in Ad-APN–treated mice increased 48 times
as much as those in Ad-ßgal treated mice. On the 14th
day after injection, the lesion formation in aortic sinus
was inhibited in Ad-APN–treated mice by 30% compared with
Ad-ßgal–treated mice (P<0.05). In the lesions
of Ad-APN–treated mice, the lipid droplets became smaller
compared with Ad-ßgal–treated mice (P<0.01).
Immunohistochemical analyses demonstrated that the
adenovirus-mediated adiponectin migrate to foam cells
in the fatty streak lesions. The real-time
quantitative polymerase chain reaction revealed that
Ad-APN treatment significantly suppressed the mRNA levels of
vascular cell adhesion molecule-1 by 29% and class A scavenger
receptor by 34%, and tended to reduce levels of tumor
necrosis factor-
without affecting those of CD36 in the
aortic tissue.
Conclusions— These findings documented for the first time that elevated plasma adiponectin suppresses the development of atherosclerosis in vivo.
Key Words: proteins • atherosclerosis
• plasma • remodeling
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Introduction |
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Adipose tissue secrets a variety of bioactive molecules that
directly contribute to the development of cardiovascular
diseases.1–4
Adiponectin is an adipose-specific plasma protein that was
identified by our group in human adipose tissue.4
Acrp30 or AdipoQ, independently cloned by 2 groups, is
the mouse counterpart of adiponectin.5,6
Interestingly, low plasma adiponectin concentrations were
observed in patients with obesity, coronary artery
disease (CAD), and type 2 diabetes with
macroangiopathy.7–9
Furthermore, the incidence of cardiovascular death was
higher in patients with low plasma adiponectin
compared with those with higher adiponectin levels.10
Immunohistochemical studies revealed that adiponectin
from the plasma adhered to the injured artery.11,12
In cultured cells, human recombinant adiponectin suppressed
the endothelial expression of adhesion molecules, the
proliferation of vascular smooth muscle cells, and the
transformation of macrophage to foam cells.8,12–14
These data suggest that adiponectin has
anti-atherogenic properties.
In the present study, we investigated the therapeutic effects of adiponectin on the development of atherosclerosis in apolipoprotein E-deficient (apoE-/-) mice.
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Method |
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Recombinant Adenovirus
Replication-defective recombinant adenovirus was constructed
with Adenovirus Expression Vector Kit (Takara). Adenovirus
expressing the full-length apM1 cDNA4
(Ad-APN) or ß galactosidase gene (Ad-ßgal) was
propagated in 293 cells, purified by CsCl gradient
centrifugations, and stored at -80°C until use.
Animal Protocol
Male apoE-/- mice (Jackson Laboratory, Bar Harbor, Me)
were fed a normal chow. At the age of 12 weeks, Ad-APN
or Ad-ßgal was injected into tail vein (2.2x108 pfu each). The mice
were anesthetized with an intraperitoneal injection of
pentobarbital (50 mg/kg), and the hearts, which
contained the aortic sinus and aortic arch, were
harvested at the indicated time. This protocol was
approved by the Institutional Laboratory Animal Care
and Use Committee of Osaka University.
Plasma Data Analyses
Blood samples were collected from mice ad libitum on the fifth
day after adenovirus injection. Total cholesterol (TC),
high-density lipoprotein cholesterol (HDL-C), and
glucose levels were measured with enzymatic kits
(Wako), and insulin levels were assayed with EIA kit
(Glazyme). Human or mouse adiponectin levels were determined
with adiponectin or Acrp30 ELISA kits (Otsuka Pharmaceutical
Co Ltd).
Histochemical Analyses
The frozen cross-sections (10 µm thick) of aortic sinus embedded
in Optimal Cutting Temperature (OCT; Sakura Finetechnical Co,
Ltd) compound after overnight fixation in 10% formalin were mounted
on slides. For the analysis of plaque size, the 3 sections (100
µm apart) from each mouse before and on the 14th day
after adenovirus injection were stained with Oil Red O. The
lesion size and the diameter of lipid droplets in the lesions
were quantified with an image analyzing computer software,
and the mean values were determined. For the
immunohistochemical analyses, frozen sections,
harvested on the fifth day after adenovirus injection,
were incubated with the either rabbit anti-human
adiponectin polyclonal antibody (OCT 9104, 2 µg/mL), rat
anti-mouse macrophages (Mac-1) monoclonal antibody (Immunotec,
20 µg/mL), or mouse anti–-smooth muscle actin (-actin) monoclonal antibody
(DAKO, 1 µg/mL). The counter-staining for the nucleus
was carried out with Mayer’s hematoxylin.
Gene Expression Analysis
To quantify expression of vascular cell adhesion molecule-1 (VCAM-1),
class A scavenger receptor (SR-A), tumor necrosis factor- (TNF-) and CD36 in aortic tissue, the real-time
quantitative polymerase chain reaction (TaqMan PCR,
Perkin-Elmer) was performed as previously described.15
The cDNA was generated from 2 µg total RNA isolated
from the upper half of the heart and aortic arch with
an oligo dT oligodeoxynucleotide primer (T12–18). The
mRNA levels of target genes were divided by those of cyclophilin
A, a standard control gene, and normalized. The values in
Ad-APN–treated aortas are expressed as a relative
percentage compared with those in Ad-ßgal treated
aortas.
Data and Statistical Analysis
Data are presented as mean±SEM and were analyzed by
Student’s t test or the Mann-Whitney U
test, depending on their distribution pattern. A value
of P<0.05 was considered statistically significant.
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Results |
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Plasma adiponectin levels in Ad-APN–treated mice increased
to a level 48 times higher than the level of endogenous
acrp30 in Ad-ßgal treated mice; however, no
significant difference was observed between 2 groups
in cholesterol, glucose, and insulin levels (Table).
In 12-week-old non-treated apoE-/- mice, the atherosclerotic
lesion area in aortic sinus and the diameter of lipid
droplets in fatty streaks were 2.98x104±0.41
µm2 and 17.8±0.2 µm, respectively. After
14 days of adenovirus injection, the lesion area of
Ad-APN–treated mice was significantly reduced by
30% compared with Ad-ßgal treated mice (5.20x104±0.62 versus 3.66x104±0.35 µm2,
P<0.05) (Figure
1A and 1B). In the lesions of Ad-APN–treated
mice, the diameter of lipid droplets in fatty streaks
was significantly reduced relative to Ad-ßgal–treated mice
(22.4±0.2 versus 18.0±0.2µm, P<0.01) (Figure
1C and 1D).
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To assess the role of elevated plasma adiponectin in
atherosclerotic lesion formation, the localization of
adenovirus-derived human adiponectin, macrophages, and
smooth muscle cells were analyzed immunohistochemically
on the fifth day after adenovirus injection. Adenovirus-derived
human adiponectin abundantly adhered to Mac-1 positive
and -actin negative cells in the
fatty streak lesions (Figure
2A).
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To further assess the mechanism of lesion reduction, the mRNA
levels of VCAM-1, SR-A, TNF-, and CD36 were quantified. Ad-APN treatment
significantly suppressed the mRNA levels of VCAM-1 (100±10.3
versus 70.9±4.8%, P<0.05) and SR-A (100±10.0
versus 65.8±9.2%, P<0.05, and tended to
reduce those of TNF- (100±33.6 versus 63.1±15.2%) without
affecting those of CD36 in the aortic tissue (100±4.5 versus
100.2±13.0%) (Figure
2B).
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Discussion |
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In the present study, we demonstrated that adenovirus-mediated
increase of plasma adiponectin significantly suppressed the
progression of atherosclerotic lesions in apoE-/-
mice. These mice develop hyperlipidemia and vascular
lesions similar to human atherosclerosis.16,17
Even on a normal chow, the initial foam cell lesions
in the aortic root progressed from 12- to 14-week-old
mice as previously reported.18
Adenovirus-derived adiponectin accumulated in the
fatty streak lesions composed of macrophages and foam
cells in apoE-/- mice. Ad-APN treatment significantly
suppressed the expression of VCAM-1 and SR-A and reduced
the lipid accumulation in macrophages in atherosclerotic lesions
of apoE-/- mice, although no difference was observed
in plasma cholesterol, glucose, and insulin levels between
Ad-APN and Ad-ßgal treatment.
We previously reported that human recombinant adiponectin
suppressed nuclear factor-
B inducible gene expression including VCAM-1
in human aortic endothelial cells and SR-A expression in
human monocyte-derived macrophages.8,12,14
The recombinant protein did not affect CD36 expression
in macrophages.12
Adiponectin treatment dose-dependently decreased the
uptake of modified low-density lipoprotein.12
In the present study, the Ad-APN treatment actually
inhibited the expression of VCAM-1 and SR-A without
affecting that of CD36 in vivo. VCAM-1 and SR-A play a
pivotal role in the development of atherosclerosis. The
expression of VCAM-1 localized over the surface of
endothelial cells in lesion-prone sites, and the
targeted disruption of SR-A reduced the size of
atherosclerotic lesions in apoE-/- mice.19,20
Therefore, our findings suggest that the elevated
plasma adiponectin protected endothelial cells from
hypercholesterolemia-induced vascular injury and
suppressed the uptake of modified low-density lipoprotein into
foam cells in apoE-/- mice.
The hypo-adiponectinemia was observed in patients with CAD and was associated with the incidence of cardiovascular death.8,10 This study documented for the first time that the overexpression of adiponectin actually reduced atherosclerosis through attenuating endothelial inflammatory response and macrophage to foam cell transformation in vivo. Therefore, in future studies, this model of adiponectin gene transfection will be useful to determine the therapeutic level of plasma adiponectin for preventing atherosclerosis.
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Acknowledgments |
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This work was supported by grants from the Japanese Ministry
of Education and the Japan Society for Promotion of
Science-Research for the Future Program.
Received August 19, 2002; revision received October 8, 2002; accepted October 8, 2002.
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