Article

Inhibition of vascular smooth muscle cell proliferation and migration in vitro and neointimal hyperplasia in vivo by adenoviral-mediated atrial natriuretic peptide delivery

Groupe Epidémiologie Clinique et Médecine, Université des Antilles et de la Guyane, Guadeloupe, France. laurent
The Journal of Gene Medicine (Impact Factor: 2.47). 07/2012; 14(7):459-67. DOI: 10.1002/jgm.2639
Source: PubMed

ABSTRACT

Vascular smooth muscle cell (VSMC) proliferation and migration are important components of the remodeling process in atherosclerosis or following angioplasty. Atrial natriuretic peptide (ANP) inhibits the growth of VSMCs in vitro but this effect has not been proven in vivo. In the present study, we examined the effects of local overexpression of ANP following gene transfer on in vitro VSMC proliferation and migration and in vivo neointimal formation in a rat carotid artery model of vascular injury.
ANP gene transfer was performed using a recombinant adenovirus containing the ANP cDNA controlled by the Rous sarcoma virus (RSV) long terminal repeat (Ad-RSV-ANP). A recombinant adenovirus expressing the RSV-controlled β-galactosidase gene (Ad-RSV-β-gal) was used as the control. Rat VSMC culture was used for in vitro studies. In the in vivo experiments, carotid arteries were analyzed after balloon injury and local infusion of the viral solution.
VSMCs transfected by Ad-RSV-ANP produced a significant amount of ANP detected by immunoreactive assay and accumulated about 6.5 times more cGMP than the viral control. VSMC proliferation stimulated with 10% fetal calf serum was reduced by 31% and migration by 25%. Fourteen days after injury, neointimal formation and the intima/media ratio were reduced by 25% and 28%, respectively, in the Ad-RSV-ANP-treated group compared to the control group.
The present study demonstrates the efficacy of recombinant adenovirus Ad-RSV-ANP with respect to inhibiting rat VSMC proliferation and migration. Our findings also provide evidence that ANP is implicated in the modulation of vascular remodeling following endothelial injury.

Full-text

Available from: Lydia Foucan
Inhibition of vascular smooth muscle cell
proliferation and migration in vitro and neointimal
hyperplasia in vivo by adenoviral-mediated atrial
natriuretic peptide delivery
Laurent Laria
1
*
,
Isabelle Déprez
2
Isabelle Pham
2
Dominique Rideau
2
Vanessa Louzier
2
Micheline Adam
3
Marc Eloit
3
Lydia Foucan
1
Serge Adnot
2
Emmanuel Teiger
2
1
Groupe Epidémiologie Clinique et
Médecine, Université des Antilles et de
la Guyane, Guadeloupe, France
2
Département de Physiologie et
INSERM U492, Faculté de médecine,
CHU Henri-Mondor, Créteil, France
3
URA INRA de Génétique Moléculaire
et Cellulaire, Génétique Virale, Ecole
Nationale Vétérinaire dAlfort,
Maisons-Alfort, France
*Correspondence to: L. Laria,
Service de Cardiologie, CHU de
Pointe-à-Pitre, 97159 Pointe-à-Pitre,
Guadeloupe, French West Indies.
E-mail: laurent laria@chu-
guadeloupe.fr; llaria@orange.fr
These investigators contributed
equally and should be considered as
senior authors.
Abstract
Background Vascular smooth muscle cell (VSMC) proliferation and migration
are important components of the remodeling process in atherosclerosis or
following angioplasty. Atrial natriuretic peptide (ANP) inhibits the growth of
VSMCs in vitro but this effect has not been proven in vivo. In the present study,
we examined the effects of local overexpression of ANP following gene transfer
on in vitro VSMC proliferation and migration and in vivo neointimal formation
in a rat carotid artery model of vascular injury.
Methods ANP gene transfer was performed using a recombinant adenovirus
containing the ANP cDNA controlled by the Rous sarcoma virus (RSV) long ter-
minal repeat (Ad-RSV-ANP). A recombinant adenovirus expressing the RSV-
controlled b-galactosidase gene (Ad-RSV- b-gal) was used as the control. Rat
VSMC culture was used for in vitro studies. In the in vivo experiments, carotid
arteries were analyzed after balloon injury and local infusion of the viral
solution.
Results VSMCs transfected by Ad-RSV-ANP produced a signicant amount of
ANP detected by immunoreactive assay and accumulated about 6.5 times more
cGMP than the viral control. VSMC proliferation stimulated with 10% fetal calf
serum was reduced by 31% and migration by 25%. Fourteen days after injury,
neointimal formation and the intima/media ratio were reduced by 25%
and 28%, respectively, in the Ad-RSV-ANP-treated group compared to the
control group.
Conclusions The present study demonstrates the efcacy of recombinant
adenovirus Ad-RSV-ANP with respect to inhibiting rat VSMC proliferation and
migration. Our ndings also provide evidence that ANP is implicated in the
modulation of vascular remodeling following endothelial injury. Copyright ©
2012 John Wiley & Sons, Ltd.
Keywords ANP; atrial natriuretic peptide; carotid artery/restenosis; gene transfer;
intimal hyperplasia; natriuretic peptide
Introduction
Migration and proliferation of vascular smooth muscle cells (VSMCs) play a major
role in the pathogenesis of atherosclerosis and restenosis. These effects are
stimulated by several growth factors, including platelet-derived growth factor
(PDGF), b roblast growth factor, insulin-like growth fact or, vasoactiv e hormones
RESEARCH ARTICLE
Received: 15 December 2011
Revised: 22 April 2012
Accepted: 24 May 2012
Copyright © 2012 John Wiley & Sons, Ltd.
THE JOURNAL OF GENE MEDICINE
J Gene Med 2012; 14: 459467.
Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jgm.2639
Page 1
(such as angiotensin, endothelin, epinephrine and serotonin)
and cytokines [1,2]. By contrast, a number of fact ors, such as
transforming growth factor-b, nitric oxide and natriuretic
peptides, act as growth inhibitors. Atrial natriuretic peptide
(ANP), brain natriuretic peptide (BNP) and C-type natriuretic
peptide (CNP) share a similar structural conformation and
some common biological actions through specic receptors
linked to particulate guanylate cyclase [3,4]. ANP and BNP
have higher afnity for the A-type natriuretic receptor
(NPR-A), whereas CNP binds to the B-type natriuretic
receptor (NPR-B) [5]. There is no w convincing evidence that
cardiac natriuretic peptides (especially ANP and CNP) are
implicated in uid homeostasis, control of vascular tone
[610] and vascular remodeling. CNP, the most studied in
this context, has been found to inhibit VSMC proliferation
and migration both in vitro [1 1] and in vivo [12]. ANP has
also been found to inhibit proliferation of VSMCs [1316],
although this effect has not been proven in vivo.Barber
et al. [17] found that infusion of ANP di rectly into the lumen
of a collared carotid artery did not reduce neointimal
hyperplasia, whereas CNP had a small effect. This absence
of an in vivo effect could reect an insufcient concentration.
CNP local delivery using a viral or non viral approach appears
to be superior to luminal administration [1 8,1 9].
Direct deliv ery of the ANP gene to endothelial cells or
VSMCs to induce overexpression of a biologically active
ANP may provide a better understanding of its local effects
and may have therapeutic relevance for diseases associated
with vascular remodeling. In previous studies, we built an
adenoviral vector containing ANP cDNA that was under
the control of the Rous sarcoma virus (RSV) 3
0
long terminal
repeat (Ad-RSV-ANP), and we found sustained increases in
plasma ANP concentration and urinary cGMP excretion in
dogs [20]. We also found that intratracheal administration
of the same adenoviral vector could reduce pulmonary
artery pressure and right ventricular hypertrophy in rats
exposed to chronic hypoxia [20]. Lin et al. [21] reported that
the delivery of a recombinant adenovirus expressing the
human ANP gene to hypertensive salt-sensitiv e Dahl rats
was associated with lower blood pressure, reduced arterial
thickening and prevention of kidney damage. However, the
effect of ANP overexpression in the vascular wall following
arterial injury has not been examined specically. In the
present study, we investigated whether an adenoviral vector
expressing ANP alters VSMC prolif eration and migration
in vitro and neointimal hyperplasia in vivo in balloon-injured
rat carotid arteries. We also investigated whether altered
NPR-A or NPR-B expression occurs during neointimal
formation.
Materials and methods
Generation of recombinant adenoviral
vectors
We have previously described the viral construction used in
the present study [20]. Briey, the canine ANP cDNA was
used in a replication-decient adenoviral vector Ad-RSV -ANP
containing an expression cassette in the E1 position with
the RSV long terminal repeat promoter/enhancer. This
vector was an E1a- and E3-deleted vector, and was obtained
by the ligation of the puried large ClaIfragmentofdl327
[22] to the NarIPstI fragment of pRSV-ANP. A recombinant
adenovirus expressing the RSV-controlled b-galactosidase
(b-gal) gene was used as a control adenoviral construction
(Ad-RSV-b-gal) [23]. Each construction was checked by
restriction analysis. The virus stocks were cloned once or
twice by plating in HEK293 cells to verify their purity. All
adenovirus vectors were propagated in HEK293 cells,
puried by CsCl gradient centrifugation, dialyzed and
stored at 80
C [24,25]. The absence of contamination
with replication-competent adenovir us was checked by the
absence of a cytopathic effect on A549 cells [26]. The ability
of the Ad-RSV-ANP to drive the synthesis of biologically
active ANP was tested in vitro in rat and human VSMCs.
Cell culture and adenovirus infection
The 293 cell line, an adenoviral-transformed human
embryonic cell line containing the E1 region of adenovirus
integrated into its genome [25], was used for adenovirus
transfection, amplication and titration. These cells were
maintained in Dulbeccosmodied Eaglesmedium
(DMEM; Gibco, Gaithersburg, MD, USA) with 10% heat-
inactivated fetal calf serum (FCS; Biomedia, Boussens,
France). The rat aortic smooth muscle cells (SMCs) were
obtained as described previously [27]. The vessel was
incubatedinDMEMsupplementedwith15U/mloftypeI
collagenase (Sigma, St Louis, MO, USA) and 2 mg/ml of
bovine serum albumin (BSA; Sigma) for 45 min at 37
C,
then dissected in DMEM supplemented with 2.6 mg/ml
BSA, 0.5 mg/ml type III elastase (Sigma) and 4.5 U/ml type
I collagenase, and incubated for 3 h at 37
C. The tissue
was then transfer red to anot her culture dish to elimi-
nate the broblasts and cultured in DMEM complemen-
ted wit h 15% heat-inactivated FCS. For the in vitro
transduction experiments, cells were seeded at a density
of 5 10
4
cells/cm
2
in DMEM supplemented with 15%
heat-inactivated FCS. The cells were used between passages
4and7.
ANP detection in culture medium
To study the production of ANP after gene transfer, a
radioimmunoassay to measure immunoreactive ANP was
performed using a (
125
I) radioimmunoassay system with
an antibody to human ANP (Amersham, Little Chalfont,
UK) characterized by marked cross-reactivity to canine
ANP. The assay was performed in accordance with the
manufacturers instructions. Rat VSMCs were plated in six-
well culture plates and infected at a multiplicity of infection
(MOI) of 50 or 500 plaque-forming units per cell (pfu/cell)
as described previously (infected cells). Cell culture
supernatants were aliquoted into tubes containing several
460 L. Laria et al.
Copyright © 2012 John Wiley & Sons, Ltd. J Gene Med 2012; 14: 459467.
DOI: 10.1002/jgm
Page 2
protease inhibitors (10 mg of ethylenediaminetetraacetic
acid, 0.1 mg of aprotinin, 0.02 mg of phenylmethylsulfonyl
uoride and 0.05 mg of trypsin inhibitor), centrifuged at
300 g for 15 min at 4
C and stored at 80
C.
Production of cGMP in culture medium
Rat carotid SMCs w ere plated in six-well plates and infected
at an MOI of 50 pfu/cell as described previously (transduced
cells). On the second postinfection day, conuent VSMCs
(nontransduced cells) were transferred into conditioned
medium. Isobutylmethylxanthine (1 0
4
M) was added to
the transduced and nontransduced cells or t o cells incubated
with ex og eno us rat ANP (10
6
M) and the cells were
incubated with 0.2% FCS. The transduced, nontransduced
and control cells were then harvested by 0.1 N HCl within
10 min at 4
C. The cGMP concentration was measured in
cell extracts using a commercial radioimmunoassay kit
(Amersham) and was normalized for protein content
measured using the Lowry method [28].
Measurement of cell growth
A tetrazolium-based nonradioactive proliferation assay was
used to measure SMC proliferation [29]. The results of this
assay correlate with
3
H-thymidine incor poration in studies
of growth factor activity. Briey, rat carotid SMCs were
seeded into 96-well tissue culture plates and incubated with
serum-free medium for 72 h to obtain quiescent nondividing
cells in the presence of Ad-RSV-ANP or Ad-RSV-b-gal at an
MOI of 0.550 pfu/cell. Thereafter, cells were incubated
with an equal volume of vehicle alone or 1 0% FCS for 72 h in
a humidied95%air/5%CO
2
atmosphere. Tetrazolium
reagents were added 4 h before the plates were read at 520
nm with an enzyme-linked immunoassay plate reader . The
experiments were conducted in quadruplicate.
Measurement of cell migration
Cell migration was quantied by measuring the migration
of cells from an agarose drop using a modication of the
method described by Planus et al.[30]andVaraniet al.
[31]. SMCs were plated for 24 h in a 75-cm
2
ask. They
were then synchronized in serum-free DMEM for 72 h and
simultaneously transduced with the adenoviral vectors
Ad-RSV-ANP or Ad-RSV-b-gal at an MOI of 2.520 pfu/cell.
After 72 h, the cells were trypsinized and resuspended at a
concentration of 40 10
6
cells/ml in DMEM containing
15% FCS and 0.3% low-melting point agarose (Sigma)
maintained at 37
C to prevent the agarose setting. Three
microliters of the cell suspension was applied to the center
of the wells within a 24-well tissue culture dish (Nunc,
Roskilde, Denmark), which was then placed at 4
Cfor
20 min to allow the agarose to solidify. The substrate used
in this experiment was poly-
DL-ornithine, and the plates
were coated with 250 mlof
DL-ornithine (12.5 mg/ml) for
5 min at room temperature, rinsed three times with sterile
water and allowed to dry before use. Agarose drops were
placed onto this matrix and covered with 1 ml of DMEM
containing 0.2% or 10% FCS after cooling. Cell migration
was measured 24 h after stimulation. In this assay, cells
migrate out to form a uniform corona around the drop. Cell
migration was quantied after digitizing the image of the
drop using image analysis software (Perfect Image, Paris,
France). For each experiment, each condition was tested
using at least 15 replicates. The mean migration was
calculated for each experiment and is expressed as a
percentage of the mean migration in the 10% FCS control
condition. No experiment was performed with an MOI
higher than 20 pfu/cell as a result of the toxic effects on
cells after transduction, trypsinization and agarose
incorporation.
Animal model and local delivery of
adenoviral construction
Male SpragueDawley rats (400500 g) were anesthetized
by intraperitoneal injection of pentobarbital (60 mg/kg)
and the neck was incised. Left common carotid de-
endothelialization was induced by the passage of a 2-F
Fogarty balloon catheter inserted into the external carotid
artery and distended with 20 ml of saline as described
previously [32]. Thirty animals were randomly divided
into three groups for the treatment with Ad-RSV-ANP,
Ad-RSV-b-gal or vehicle. The adenoviral solution or
phosphate-buffered saline (PBS) was introduced into the
left common carotid artery. The adenoviral solution
(2.5 10
8
pfu, 50 ml) or PBS was introduced into the left
common carotid artery and kept in contact for 30 min by
clamping. The neck wound was repaired and the animal
was allowed to recover. Two weeks after the carotid
balloon injury, the rats were killed and in situ pressure
perfusion xation was performed using 4% formaldehyde
as descr ibed previously [33]. The left common carotid
arteries were excised, placed in the same xative for
24 h, and then embedded in parafn. The proximal half
of each artery was selected for histological analysis.
Quadruplicate slides of 5-mm sections were stained with
orcein-picroindigocarmine. Morphometric analysis was
perfor med with a computerized digital microscopic
algorithm (Perfect Image, Paris, France) on cross-sectional
areas of media, intima and lumen under blind conditions.
The animal care complied with the Guide for the Care
and Use of Laboratory Animals.
In vivo expression of ANP in the carotid
artery after local delivery of adenoviral
construction
W e used immunochemistry to detect ANP synthesis in the
carotid wall. Briey, previously injured caroti d arteries
treated with Ad-RSV -ANP, Ad-RSV-b-gal or PBS were xed
in Bouins solution. Endogenous peroxidase activity was
inhibited using 0.6% hydrogen peroxide in methanol, and
ANP gene delivery in rat carotid artery 461
Copyright © 2012 John Wiley & Sons, Ltd. J Gene Med 2012; 14: 459467.
DOI: 10.1002/jgm
Page 3
nonspecic binding sites were blocked with normal goat
serum. The slides were incubated overnight at 4
Cwitha
1:1000 dilution of specic polyclonal antibody against human
ANP 128 (a gift from Dr D. Springall, Royal Postgraduate
Medical School, Hammersmith Hospital, London , UK),
which has 100% cross-reactivity with the canine ANP 128.
Control slides w ere treated with normal dilute rabbit serum.
Samples were incubated with a biotinylated goat anti-rabbit
antibody and a horseradish peroxidase-labeled streptavidin
solution (Dako, Glostrup, Denmark). Staining was obtained
after 5 min of incubation in 3,3
0
-diaminobenzidine(Sigma)
to visuali ze the peroxida se activity.
Reverse transcr iption-polymerase chain
reaction (RT-PCR) analysis of NPR-A and
NPR-B expression in the carotid artery
after arterial injury
For RT of extracted mRNA, 3 mg of total RNA was denatured
for 5 min at 65
C. The samples were cooled, and the
following components were added: 9 mlof5 RT buffer,
3 ml of 2.5 mmol/l deoxynucleotide mixture, 1.5 mlof
0.5 mg/ml oligo-dT primer, 1.5 ml of 0.1 mol/l dithiothreitol
and 3 ml of Moloney leukemia virus reverse transcriptase
(Gibco). After 60 min of incubation at 37
C, the reverse
transcriptase was inactivated by heating the mixture at
65
C for 10 min. For PCR, 5 ml of the RT reaction was added
to 5 mlof10mmol/l forward primer, 5 mlof10mmol/l
deoxynucleotide mixture, 3 mlof50mmol/lMgCl
2
,10mlof
10 amplication buffer, 6 mlofH
2
O and 0.5 mlofTaq
polymerase (Gibco). The thermal cycler program com-
prised an initial incubation at 94
C for 3 min followed by
40 cycles (93
C for 60 s, 52
Cfor120sand72
Cfor180s)
and a nal extension at 72
Cfor10min.
The receptor primers for rat NPR-A, NPR-B and NPR-C
were designed from published sequences [34]: for NPR-A
forward, 5
0
-GAC TTG CAG CCC AGC AGC CTG-3
0
and
reverse, 5
0
-CAG GTG GTC CTG CAG ATC CAT-3
0
(predicted
length of the PCR product, 780 bp); for NPR-B forward, 5
0
-
TCA AAC ACA TGA GAG ATG TTC-3
0
and reverse, 5
0
-TAT
TGG CAT ACT GTT CCA TGC-3
0
(predicted length of the
PCR product, 716 bp); and for NPR-C forward, 5
0
-CGA
CCG GGA GAG AGA GGC-3
0
and reverse, 5
0
-CAG AAC TTT
TCA CCT CCA TGG-3
0
(predicted length of the PCR prod-
uct, 903 bp). Amplied DNA was size-fractionated by elec-
trophoresis on agarose gels containing ethidium bromide.
Negative controls included the omission of the RT reaction
and amplication in the absence of templates.
Statistical analysis
The results are expressed as the mean SD. Analysis of
variance was used for comparison between groups for the
in vitro and the in vivo experiments. Bonferroni post-hoc
tests were performed for comparison 2 by 2. SPSS, version
19.0 (SPSS Inc., Chicago, IL, USA) was used for data
analysis. p < 0.05 (two-sided) was considered statistically
signicant.
Results
ANP secretion and cGMP production by
Ad-RSV-ANP-transduced SMCs
Rat VSMCs transfected with Ad-RSV-ANP at MOIs of 50
and 500 pfu/cell accumulated immunoreactive ANP in
the cell medium starting on the rst postinfection day.
The rate of immunoreactive ANP production was related
to the MOI. After 7 days, the amount of immunoreactive
ANP in the conditioned medium reached 97 46 pg/10
6
cells at an MOI of 50 pfu/cell and 1229 622 pg/10
6
cells
at an MOI of 500 pfu/cell (Figure 1). Immunoreactive
ANP was undetectable in media from nontrans duced cells
and from Ad-RSV-b-gal-infected cells.
To determine whether the transduced ANP gene encoded
a biologically active protein, we assessed cGMP production
in VSMCs transduced with Ad-RSV-ANP or Ad-RSV-b-gal at
an MOI of 500 pfu/cell. As shown in Figure 2, cells
transduced with Ad-RSV-ANP produced approximately
6.5 times as much cGMP compared to Ad-RSV-b-gal-
transduced cells (p < 0.01). Cells incubated with condi-
tioned medium from cells transduced with Ad-RSV-ANP at
the same MOI also accumulated at least 3.5 times the
amount of cGMP as in the viral control, Ad-RSV-b-gal
(p < 0.01). Incubation of VSMCs with ANP (10
6
M)also
caused a signicant and similar accumulation of intracellu-
lar cGMP: four times compared to cells incubated with
0.2% FCS (p < 0.005).
Effects of Ad-RSV-ANP on SMC
proliferation
Cell proliferation was stimulated by 47% in the presence of
medium containing 10% FCS compared to cells cultured in
Figure 1. ANP secretion by VSCM transduced with Ad-RSV-ANP
at an MOI of 50 and 500 pfu. ANP secretion was undetectable
in media from nontransduced cells and cells transfected with
control vector (Ad-RSV-b-gal)
462 L. Laria et al.
Copyright © 2012 John Wiley & Sons, Ltd. J Gene Med 2012; 14: 459467.
DOI: 10.1002/jgm
Page 4
the presence of 0.2% FCS (Figure 3). In cells infected with
Ad-RSV-ANP at an MOI of 50 pfu/cell, the increment of
growth response to FCS was reduced by 16% (p < 0.05)
and 31% (p < 0.001) on days 3 and 4, respectively. A lower
MOI (0.5 and 5 pfu/cell) had no effect on cell proliferation.
No signicant effect was observed with Ad-RSV-b-gal-
infected cells even at an MOI of 50 pfu/cell.
Effects of Ad-RSV-ANP and exogenous
ANP on SMC migration
Cell migration from agarose drop explants was stimulated
1.7-fold in the presence of medium complemented with
10% FCS compared to that measured in the presence of
0.2% FCS (data not shown). As illustrated in Figure 4, pre-
infection of SMCs with Ad-RSV-ANP at an MOI of 20 pfu/cell
inhibited cell migration by 25 11% (p < 0.005), whereas
treatmentwithAd-RSV-ANPatlowerMOIsof2.5and5
pfu/cell had no effect. No change in cell migration was
observed in cells pre-infected with Ad-RSV-b-gal. A similar
inhibition of migration (26%) was observed when the
agarose drop was incubated in medium containing 10
6
M
ANPbutnotatalowerconcentration.
Effects of intraluminal administration
of Ad-RS V-ANP on neointimal formation
after balloon-induced carotid artery
injury
Detectable amounts of ANP were found on day 5 following
Ad-RSV-ANP treatment (Figure 5), although no ANP was
detectable in arteries treated with Ad-RSV-b-gal or PBS.
Neointimal formation occurred within 14 days after carotid
injury (Figure 6). As indicated in Table 1, the neointimal
area was reduced by 25% in the Ad-RSV-ANP-treated group
(0.113 0.021 mm
2
; p < 0.01) compared to the PBS-
treated (0.150 0.030 mm
2
) and Ad-RSV-b-gal-treated
(0.149 0.035 mm
2
) groups. The intima/media ratio was
reduced by 28% in the Ad-RSV-ANP-treated group
(0.96 0.12; p < 0.001) compared to the Ad-RSV-b-gal-
treated (1.27 0.23) and PBS-treated (1.34 0.18)
groups. The neointimal area did not differ signicantly be-
tween the two control groups. The inhibition of neointimal
hyperplasia by Ad-RSV-ANP was limited to the treated
(proximal half) segment of the carotid artery (data not
shown). The media area did not differ between the three
groups (PBS-treated group: 0.113 0.024 mm
2
;Ad-RSV-
b-gal-treated group: 0.117 0.021 mm
2
;Ad-RSV-ANP-
treated group: 0.117 0.014 mm
2
; p = 0.89). The lumen
area did not differ between the three groups (p =0.95).
Expression of ANP receptors as assessed
by RT-PCR
RT-PCR products showed that NPR-A and NPR-B were
expressed in both intact and injured arteries. The expression
of these two receptors was not modulated 4 days after
injury (Figure 7).
Discussion
In the present study, adenoviral-mediated ANP overexpres-
sion in carotid SMCs in culture medium inhibited both
Figure 2. cGMP production (ratio to the control) by aortic SMCs
transduced with Ad-RSV-ANP at an MOI of 500 pfu/cell and in the
culture medium from transduced cells. Comparison with Ad-RSV-
b-gal-infected SMCs. *p < 0.01; **p < 0.005; p < 0.01
Figure 3. Effect of Ad-RSV-ANP at MOIs of 5 and 50 pfu/cell on VSMC proliferation. Signicant differences from t he control: *p < 0.05
and p < 0.001
ANP gene delivery in rat carotid artery 463
Copyright © 2012 John Wiley & Sons, Ltd. J Gene Med 2012; 14: 459467.
DOI: 10.1002/jgm
Page 5
proliferation (25%) and migration (26%). In our in vivo
rat carotid artery model of intimal injury, the adenoviral
ANP vector was associated with a 25% reduced neointimal
area and 28% reduced intima/media ratio. To our
knowledge, this is the rst study to show the in vivo effects
of ANP adenoviral delivery on arterial remodeling after
vascular injury. We found that NPR-A and NPR-B were still
expressed in the SMCs 14 days after vascular injury.
Cultured VSMCs infected with Ad-RSV-ANP but not with
Ad-RSV-b-gal accumulated ANP in cell culture supernatants
within 48 h after transduction. This production of ANP
correlated with the production of cGMP, showing that the
newly-expressed ANP was biologically active and acted as
a paracrine factor. This increase in cGMP level was not
caused by activation of nitric oxide synthase because nitrite
and nitrate levels did not differ between the medium
from control cells and cells infected with Ad-RSV-ANP or
Ad-RSV-b-gal (data not shown).
Previous studies found that ANP exerts an antimitogenic
effect in rat mesangial cells [14,35,36]. Abell et al.[13]
found that ANP had little effect on basal rat aor tic SMC
growth but dose-dependently inhibited PDGF-stimulated
thymidine incorporation. The increase in DNA synthesis
was reduced by almost 25% and 60% by 10
6
and 10
7
M
ANP, respectively. In the present study, treatment of VSMCs
in culture medium supplemented with 10% FCS reduced
the cell proliferation at the highest MOI of 50 pfu/cell.
Similar effects were observed by Morishita et al.[16]on
SMC and endothelial cell proliferation using either a
liposomal vector of rat ANP or exogenous ANP.
In addition to its effect on SMC growth, Ad-RSV-ANP
strongly inhibited vascular FCS-stimulated SMC migration.
The migratory capacity of SMCs was also reduced markedly
in response to 1 0
6
M exogenous ANP, suggesting that ANP is
equally effectiv e at inhibiting vascular SMC migration and
proliferation. This result is consistent with previous studies
showing that ANP inhibits the migration of FCS-, PDGF- or
oxidized low-density lipoprotein-stimulated vascular SMCs
in a concentration-dependent manner [3 7,38].
In vivo application of Ad-RSV-ANP into the balloon-
injured rat carotid artery signicantly reduced neointimal
formation by almost 30%. This nding is consistent with
the fact that neointimal formation seen after percutaneous
transluminal angioplasty reects the combination of SMC
proliferation and migration from the media to the intima.
This nding also conrmed in vivo the role of ANP in the
Figure 4. Effect of Ad-RSV-ANP on SMC migration from agarose drop explants af ter stimulation with 10% FCS. A marked inhibition of
migration was observed in treated cells compared to controls. *p < 0.01. This inhibition was similar to that caused by exogenous ANP
at 10
6
M
Figure 5. In vivo expression of ANP in the carotid artery after local delivery of t he adenoviral construct. Immunostaining with an
antibody specic to canine ANP in cross-sections of balloon-injured rat carotid artery (5 days after injury). Strong immunoreactivity
for ANP was present in the neointimal layer (arrows) of the Ad-RSV-ANP-treated group, whereas no reaction was observed in the PBS
and Ad-RSV-b-gal control groups
464 L. Laria et al.
Copyright © 2012 John Wiley & Sons, Ltd. J Gene Med 2012; 14: 459467.
DOI: 10.1002/jgm
Page 6
remodeling process after vascular injury. These results are
similar to those obtained by Ueno et al. [39] with another
natriuretic peptide, which showed that local expression of
CNP inhibits neointimal formation in rat injured arteries.
In the latter study, treatment of cultured rat SMCs with an
adenoviral construct encoding CNP was associated with
secretion of the peptide and moderate inhibitory effects on
SMC proliferation but with a 90% reduction in neointimal
formation. Furuya et al. [12] demonstrated that constan t
intravenous administration of CNP for 5 or 14 days reduced
neointimal formation by 60% to 70% in a similar model,
compared to 35% in the present study. Although the
experimental conditions were not identical, these ndings
suggestthatCNPisamorepotentinhibitorofin vivo
neointimal hyperplasia than ANP, as sugge sted by previous
in vitro studies [40]. These differences might reec t the
different expression levels of the respective NPR-B and
NPR-A. NPR-A is expressed predominantly at the mRNA
Figure 6. Effects of Ad-RSV-ANP on neointimal formation after arterial injury. Light microscopy of representative cross-sectioned
carotid ar teries 14 days after injury. The images show a comparison between arteries obtained from rats treated with Ad-RSV-ANP
and PBS and Ad-RSV-b-gal controls. Morphometric analysis showed a signicant (p < 0.005) difference in the treated group compared
to the control groups. NI, neointima; M, media
Table 1. Morphometric analysis of rat carotid arteries 14 days after injury
Cross-sectional area 1000 mm
2
Intima/media
ratioIntima Media Lumen
Ad-RSV-ANP (n = 8) 113 21* 117 14 322 82 0.96 0.12**
Ad-RSV-b-gal (n = 8) 149 35 117 21 321 105 1.27 0.23
PBS (n = 8) 150 30 113 24 308 107 1.34 0.18
Data are expressed as the mean SD; n, number of rats in each group. Analysis shows a signicant reduction of neointimal area
(*p < 0.01) and intima/media ratio (**p < 0.001) in the Ad-RSV-ANP-treated group compared to the PBS control group.
Figure 7. RT-PCR analysis of NPR-A and NPR-B in intact (lanes 1
and 3, n = 3) and injured (lanes 2 and 4, n = 3) carotid arteries.
The comparison was performed on days 5 (lane 1 versus 2) and
14 (lane 3 versus 4). GAPDH, glyceraldehyde-3-phosphate
dehydrogenase
ANP gene delivery in rat carotid artery 465
Copyright © 2012 John Wiley & Sons, Ltd. J Gene Med 2012; 14: 459467.
DOI: 10.1002/jgm
Page 7
level in intact rat aortic SMCs, whereas NPR-B is expressed
predominantly in cultured cells [41]. It can be hypothesized
that the more potent action of CNP relates to the phenotype-
related expression of natriuretic receptors. However, NPR-A
and NPR-B mRNA expression measured by RT-PCR did not
differ betw een injured and intact rat carotid arteries. These
results do not support the concept that ANP NPR-A
markedly decreases after de-endothelialization, as reported
previously in a rabbit model of injury caused by compression
ofthecentralearartery[42].Thisdifferencemayreect
differences in the animal model or experimental conditions.
An alternative explanation for the more potent action of
CNP may be the higher binding afnity of the clearance
receptor NPR-C to ANP, as demonstrated by Suga et al.
[41]. It is possible that, in the present study, the in vivo
effects of ANP were limited by its short half-life.
Furthermore, a complete re-endothelialization was
observed 2 weeks after injury in all Ad-RSV-ANP treated
arteries, as well as in control groups, suggesting that inhibi-
tion of neointima formation by this approach did not com-
promise endothelial repair. This nding could have an inter-
est in the prevention of restenosis or stent thrombosis
following angioplasty.
In summary, the ndings of the present study demon-
strate that the recombinant adenovirus Ad-RSV-ANP is
an effective tool for modulating SMC proliferation and
migration. The results obtained in the de-endothelialized
rat carotid arter y provide evidence that ANP plays a
protective role against changes in proliferation and
migration in vivo in the injured artery. Adenovirus-mediated
local expression of ANP has potential as an effective form
of molecular intervention in proliferative arterial diseases.
Acknowledgements
The authors declare that there are no conicts of interest.
References
1. Dzau VJ, Gibbons GH, Cooke JP, et al.
V a scular biology and medicin e in the
1 990s: scope, concepts, potentials, and
perspectives. Circulation 1993; 87 :705719.
2. Thyberg J, Hedin U, Sjolund M, et al.
Regulation of differentiated properties
and proliferation of arterial smooth
muscle cells. Arteriosclerosis 1990; 10:
966990.
3. Anand-Srivastava MB, Trachte GJ. Atrial
natriuretic factor receptors and signal
transduction mechanisms. Pharmacol
Rev 1993; 45: 455497.
4. Pandey KN. Natriuretic peptides and their
receptors. Peptides 2005; 26:899900.
5. Suga S, Nakao K, Hosoda K, et al. Recept o r
selectivity of natriuretic peptide family,
atrial natriuretic peptide, brain natriuretic
peptide, and C-type natriuretic peptide.
Endocrinology 1992; 130: 229239.
6. Clerico A, Emdin M. Diagnostic accuracy
and prognostic relevance of the mea-
surement of cardiac natriuretic peptides:
a review. Clin Chem 2004; 50:3350.
7. Cody RJ, Atlas SA, Laragh JH. Physio-
logic and pharmacologic studies of atrial
natriuretic factor: a natriuretic and
vasoactive peptide. J Clin Pharmacol
1987; 27:927936.
8. Edwards BS, Ackermann DM, Schwab
TR, et al. The relationship between
atrial granularity and circulating atrial
natriuretic peptide in hamsters with
congestive heart failure. Mayo Clin Proc
1986; 61:517521.
9. McGrath MF, de Bold AJ. Determinants
of natriuretic peptide gene expression.
Peptides 2005; 26: 933943.
10. McGrath MF, de Bold ML, de Bold AJ. The
endocrine function of the heart. Trends
Endocrinol Metab 2005; 16:469477 .
11. Brown C, Pan X, Hassid A. Nitric oxide
and C-type atrial natriuretic peptide
stimulate primary aortic smooth muscle
cell migration via a cGMP-dependent
mechanism: relationship to microla-
ment dissociation and altered cell
morphology. Circ Res 1999; 84:655667.
12. Furuya M, Aisaka K, Miyazaki T, et al.
C-type natriuretic peptide inhibits
intimal thickening after vascular injury.
Biochem Biophys Res Commun 1993;
193: 248253.
13. Abell TJ, Richards AM, Ikram H, et al.
Atrial natriuretic factor inhibits prolifer-
ation of vascular smooth muscle cells
stimulated by platelet-derived growth
factor. Biochem Biophys Res Commun
1989; 160: 13921396.
14. Appel RG. Growth inhibitory activity of
atrial natriuretic factor in rat glomerular
mesangial cells. FEBS Lett 1988; 238:
135138.
15. Itoh H, Pratt RE, Dzau VJ. Atrial natri-
uretic polypeptide inhibits hypertrophy
of vascular smooth muscle cells. J Clin
Invest 1990; 86: 16901697.
16. Morishita R, Gibbons GH, Pratt RE, et al.
Autocrine and paracrine effects of atrial
natriuretic peptide gene transfer on
vascular smooth muscle and endothelial
cellular growth. J Clin Invest 1994; 94:
824829.
17. Barber MN, Gaspari TA, Kairuz EM, et al.
Atrial natriuretic peptide preserves
endothelial function during intimal
hyperplasia. J Vasc Res 2005; 42:
101110.
18. Pelisek J, Fuchs AT, Kuehnl A, et al.
C-type natriuretic peptide for reduction
of restenosis: gene transfer is superior
over single peptide administration.
J Gene Med 2006; 8: 835844.
19. Yasuda S, Kanna M, Sakuragi S, et al.
Local delivery of single low-dose of
C-type natriuretic peptide, an endoge-
nous vascular modulator, inhibits neointi-
mal hyperplasia in a balloon-injured rab-
bit iliac artery model. J Cardiovasc
Pharmacol 2002; 39:784788.
20. Chetboul V, Adam M, Deprez I, et al
.
Expression of biologically active atrial
natriuretic factor following intrahepatic
injection of a replication-defective adeno-
viral vector in dogs. Hum Gene Ther 1999;
10:281290.
21. Lin KF, Chao J, Chao L. Atrial natriuretic
peptide gene delivery attenuates hyper-
tension, cardiac hypertrophy, and renal
injury in salt-sensitive rats. Hum Gene
Ther 1998; 9: 14291438.
22. Shenk T, Williams J. Genetic analysis of
adenoviruses. Curr Top Microbiol Immunol
1 984; 111:139.
23. Oualikene W, Gonin P, Eloit M. Lack of
evidence of phenotypic complementation
of E1A/E1B-deleted adenovirus type 5
upon superinfection by wild-type virus
in the cotton rat. J Virol 1995; 69:
65186524.
24. Eloit M, Gilardi-Hebenstreit P, Toma B,
et al. Construction of a defective adeno-
virus vector expressing the pseudorabies
virus glycoprotein gp50 and its use as a
live vaccine. J Gen Virol 1990; 71:
24252431.
25. Graham FL, Smiley J, Russell WC, et al.
Characteristics of a human cell line
transformed by DNA from human
adenovirus type 5. J Gen Virol 1977;
36:5974.
26. Dion LD, Fang J, Gar ver RI Jr. Superna-
tant rescue assay vs. polymerase chain
reaction for detection of wild type ade-
novirus-contaminating recombinant ad-
enovirus stocks. J Virol Methods 1996;
56:99107 .
27. Chamley JH, Campbell GR, McConnell
JD, et al. Comparison of vascular smooth
muscle cells from adult human, monkey
and rabbit in primary culture and in
subculture. Cell Tissue Res 1977; 177:
503522.
28. Lowry OH, Rosebrough NJ, Farr AL,
et al. Protein measurement with the
466 L. Laria et al.
Copyright © 2012 John Wiley & Sons, Ltd. J Gene Med 2012; 14: 459467.
DOI: 10.1002/jgm
Page 8
Folin phenol reagent. J Biol Chem 1951;
193: 265275.
29. Mosmann T. Rapid colorimetric assay
for cellular growth and survival: appli-
cation to proliferation and cytotoxicity
assays. J Immunol Methods 1983; 65:
5563.
30. Planus E, Galiacy S, Matthay M, et al.
Role of collagenase in mediating
in vitro alveolar epithelial wound repair.
J Cell Sci 1999; 112: 243252.
31. Varani J, Orr W, Ward PA. A comparison
of the migration patterns of normal and
malignant cells in two assay systems.
Am J Pathol 1978; 90:159172.
32. Clowes AW, Reidy MA, Clowes MM.
Kinetics of cellular proliferation after
arterial injury. I. Smooth muscle growth
in the absence of endothelium. Lab Invest
1983; 49:327333.
33. Villa AE, Guzman LA, Poptic EJ, et al.
Effects of antisense c-myb oligonucleo-
tides on vascular smooth muscle cell
proliferation and response to vessel wall
injury. Circ Res 1995; 76:505513.
34. Lin X, Hanze J, Heese F, et al.Geneexpres-
sion of natriuretic peptide receptors in myo-
cardial cells. Circ Res 1 995; 77:750758.
35. Johnson A, Lermioglu F, Garg UC, et al.A
novel biological effect of atrial natriuretic
hormone: inhibition of mesangial cell
mitogenesis. Biochem Biophys Res
Commun 1988; 152:893897.
36. Kohno M, Ikeda M, Johchi M, et al.Inter-
action of PDGF and natriuretic peptides
on mesangial cell proliferation and
endothelin secretion. Am J Physiol 1993;
265:E673E679.
37. Ikeda M, Kohno M, Yasunari K, et al.
Natriuretic peptide family as a novel
antimigration factor of vascular smooth
muscle cells. Arterioscler Thromb Vasc
Biol 1997; 17:731736.
38. Kohno M, Yokokawa K, Yasunari K,
et al.
Effect of natriuretic peptide family on
the oxidized LDL-induced migration of
human coronary artery smooth muscle
cells. Circ Res 1997; 81: 585590.
39. Ueno H, Haruno A, Morisaki N, et al.
Local expression of C-type natriuretic
peptide markedly suppresses neointimal
formation in rat injured arteries through
an autocrine/paracrine loop. Circulation
1997; 96:22722279.
40. Furuya M, Takehisa M, Minamitake Y,
et al. Novel natriuretic peptide, CNP,
potently stimulates cyclic GMP production
in rat cultured vascular smooth muscle
cells. Biochem Biophys Res Commun
1 990; 170:201208.
41. Suga S, Nakao K, Kishimoto I, et al.
Phenotype-related alteration in expression
of natriuretic peptide receptors in aortic
smooth muscle cells. Circ Res 1992;
71:3439.
42. Brown J, Chen Q. Regional expression of
natriuretic peptide receptors during the
formation of arterial neointima in the
rabbit. Circ Res 1995; 77: 906918.
ANP gene delivery in rat carotid artery 467
Copyright © 2012 John Wiley & Sons, Ltd. J Gene Med 2012; 14: 459467.
DOI: 10.1002/jgm
Page 9
  • Source
    [Show abstract] [Hide abstract] ABSTRACT: Atrial natriuretic peptide (ANP) plays a pivotal role in modulation of vascular function and it is also involved in the pathophysiology of several cardiovascular diseases. We provide an updated overview of the current appraisal of ANP vascular effects in both animal models and humans. We describe the physiological implications of ANP vasomodulatory properties as well as the involvement of ANP, through its control of vascular function, in hypertension and heart failure. The principal molecular mechanisms underlying regulation of vascular tone, that is natriuretic peptide receptor type A/cyclic guanylate monophosphate, natriuretic peptide receptor type C, nitric oxide system, are discussed. We review the literature on therapeutic implications of ANP in hypertension and heart failure, examining the potential use of ANP analogues, neutral endopeptidase (NEP) inhibitors, ACE/NEP inhibitors, angiotensin receptor blocker (ARB)/NEP inhibitors, the new dual endothelin-converting enzyme (ECE)/NEP inhibitors and ANP-based gene therapy. The data discussed support the role of ANP in different pathological conditions through its vasomodulatory properties. They also indicate that ANP may represent an optimal therapeutic agent in cardiovascular diseases.
    Full-text · Article · Mar 2013 · Journal of Hypertension
  • [Show abstract] [Hide abstract] ABSTRACT: Endothelial cell (EC) activation and inflammation is a key step in the initiation and progression of many cardiovascular diseases. Targeted delivery of therapeutic reagents to inflamed EC using nanoparticles is challenging as nanoparticles do not arrest on EC efficiently under high shear stress. In this study, we developed a novel polymeric platelet-mimicking nanoparticle for strong particle adhesion onto ECs and enhanced particle internalization by ECs. This nanoparticle was encapsulated with dexamethasone as the anti-inflammatory drug, and conjugated with polyethylene glycol, glycoprotein 1b, and trans-activating transcriptional peptide. The multi-ligand nanoparticle showed significantly greater adhesion on P-selectin, von Willebrand Factor, than the unmodified particles, and activated EC in vitro under both static and flow conditions. Treatment of injured rat carotid arteries with these multi-ligand nanoparticles suppressed neointimal stenosis more than unconjugated nanoparticles did. These results indicate that this novel multi-ligand nanoparticle is efficient to target inflamed EC and inhibit inflammation and subsequent stenosis.
    No preview · Article · May 2013 · Journal of Cardiovascular Translational Research
  • [Show abstract] [Hide abstract] ABSTRACT: Stem cells are characterized by their ability to differentiate into multiple cell lineages and display the paracrine effect. The aim of this work was to evaluate the effect of therapy with bone marrow cells (BMCs) on blood glucose, lipid metabolism and aortic wall remodeling in mice through the administration of a high fat diet and subsequent BMCs transplantation. C57BL/6 mice were fed a control diet (CO group) or an atherogenic diet (AT group). After 16 weeks, the AT group was divided into four groups: an AT 14 days group and AT 21 days group, that were given an injection of vehicle and sacrificed at 14 and 21 days after, respectively; AT-BMC 14 days group and AT-BMC 21 days group that was given an injection of BMCs and sacrificed at 14 and 21 days after. The CO group was sacrificed along with other groups. The BMCs transplant had reduced blood glucose, triglycerides and total cholesterol. The Qa (1/mm(2)) was quantitatively reduced in AT 14 days group, AT 21 days group and was high in AT-BMC 21 days group. The AT 21 days group exhibited increased tunica media and elastic system fibers. The immunolabeling for α-SMA and VEGF showed less immunolabeling in transplanted groups with BMCs. The immunostaining for PCNA seems to be more expressive in the group AT-BMC 21 days group. To conclude, our results support the concept that in mice, the injection of BMCs improve glucose levels, lipid metabolism and remodeling of the aortic wall in animals using atherogenic diet.
    No preview · Article · Oct 2014 · International journal of clinical and experimental pathology
Show more