© 1999 American Heart Association, Inc.
Original Contributions |
Prostacyclin Synthase Gene Transfer Accelerates Reendothelialization and Inhibits Neointimal Formation in Rat Carotid Arteries After Balloon Injury
From the Department of Internal Medicine II (Y.N., M.H., H.O., S.M., K.M., H.M., Y.T., K.O., T.H.) and Physiology (K.N.), Nagoya University School of Medicine, and Nagoya University School of Health Science (T.I.), Department of Internal Medicine II, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.
Correspondence to Yasushi Numaguchi, Department of Internal Medicine II, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan. E-mail numa2{at}tsuru.med.nagoya-u.ac.jp
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Abstract—Prostacyclin (PGI2), a metabolite of arachidonic acid, has the vasoprotective effects of vasodilation, anti-platelet aggregation, and inhibition of smooth muscle cell proliferation. We hypothesized that an overexpression of endogenous PGI2 may accelerate the recovery from endothelial damage and inhibit neointimal formation in the injured artery. To test this hypothesis, we investigated in vivo transfer of the PGI2 synthase (PCS) gene into balloon-injured rat carotid arteries by a nonviral lipotransfection method. Seven days after transfection, a significant regeneration of endothelium was observed in the arteries transfected with a plasmid carrying the rat PCS gene (pCMV-PCS), but little regeneration was seen in those with the control plasmid carrying the lacZ gene (pCMV-lacZ) (percent luminal circumference lined by newly regenerated endothelium: 87.1±6.9% in pCMV-PCS-transfected vessels and 6.9±0.2% in pCMV-lacZ vessels, P<0.001). BrdU staining of arterial segments demonstrated a significantly lower incorporation in pCMV-PCS-transfected vessels (7.5±0.3% positive nuclei in vessel cells) than in pCMV-lacZ (50.7±9.6%, P<0.01). Moreover, 2 weeks after transfection, the PCS gene transfer resulted in a significant inhibition of neointimal formation (88% reduction in ratio of intima/media areas), whereas medial area was similar among the groups. Arterial segments transfected with pCMV-PCS produced significantly higher levels of 6-keto-PGF1
, the
main metabolite of PGI2, compared with the segments
transfected with pCMV-lacZ (10.2±0.55 and 2.1±0.32 ng/mg tissue for
pCMV-PCS and pCMV-placZ, P<0.001). In conclusion, this
study demonstrated that an in vivo PCS gene transfer increased the
production of PGI2 and markedly inhibited
neointimal formation with accelerated
reendothelialization in rat carotid arteries after
balloon injury.
Key Words: prostacyclin • prostacyclin synthase • restenosis • gene therapy • balloon injury
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Introduction |
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Vascular diseases are main life-threatening complexes in western countries.1 Arterial interventional therapy such as balloon angioplasty and stenting is presently a potential strategy against coronary artery disease.1 2 3 4 However, the efficacy of these techniques is limited by restenosis (ie, the recurrence of the treated lesions, in 20% to 50% of procedures).1 2 3 4 Although some pharmacological agents5 6 7 8 and devices9 10 11 have been shown to decrease the incidence of restenosis, the results are not sufficient and other novel therapies are required for the further decrease or elimination of restenosis.
Intensive studies of gene therapy against restenosis have been performed and have demonstrated its superior efficacy in some animal models.12 13 14 15 16 17 18 19 20 21 22 23 In these studies, the strategies for reducing restenosis may be categorized into 3 groups according to the processes: cytostatic strategies, which focus on the inhibition of cell cycle entry (eg, retinoblastoma protein RB,12 cyclinkinase inhibitor protein p21,13 tissue inhibitor of matrix metalloproteinase-114 15 ); cytotoxic strategies, which induce the death of cells that have entered the cell cycle (eg, thymidine kinase isozyme derived from herpes simplex virus,16 cytosine deaminase17 ); and paracrine strategies, which affect cells at several stages in the progression of restenosis, such as antithrombosis, reendothelialization, and the inhibition of smooth muscle cell proliferation (eg, hirudin,18 c-type natriuretic peptide,19 endothelial nitric oxide synthase,20 21 prostaglandin H synthase-1,22 vascular endothelium growth factor23 ). In a paracrine mechanism, a secreted molecule could exert effects on uninfected cells around infected cells, and more efficient inhibitory effects might thus be achieved. Moreover, a multifactorial molecule would be more effective for preventing restenosis than a simple SMC growth inhibition, because complicated mechanisms underlie the process of restenosis.
Prostacyclin (PGI2),24 25 an arachidonic acid metabolite, binds to PGI2 receptor, which induces the production of cAMP.24 PGI2 exerts its multiple effects mostly by the production of cAMP, such as vasodilation,26 anti-platelet aggregation,24 25 26 27 inhibition of smooth muscle proliferation,28 and the modulation of cholesterol turnover.29 PGI2 may be involved in many events in the regulation of vascular tone and growth through these multiple effects. It was postulated that the barrier function of the endothelium is disrupted after balloon injury, and adhesion of platelets and release of their growth factors may induce proliferative changes and result in restenosis.29 30 31 32 33 34 The earlier that endothelial function could be recovered, the greater reduction of restenosis would be gained. To test our hypothesis that an overexpression of endogenous PGI2 may accelerate the recovery from endothelial damage and inhibit neointimal formation in the injured artery, and to investigate the pathophysiological significance of endogenous PGI2, we constructed a plasmid expressing PCS. In the present study, using a balloon-injured rat artery model, we examined whether the local delivery of the PCS gene could prevent proliferative changes without inducing systemic side effects.
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Methods |
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Plasmid DNA Preparation
Based on the obtained sequence of the rat PCS gene,35 we designed the primers (forward primer, 5'-CATGTTCTGGGCCGCT-3'; reverse primer, 5'-GGAACAACAGGAAGTCAGGAG-3'; underlined region indicates starting codon). Total cellular RNA was extracted from rat aorta by the modified method of Chomczinski and Sacchi,36 and subjected to reverse-transcription using a first-strand cDNA synthesis kit (Pharmacia Biotech). In each tube, oligo(dT)12 to 18 primer (Invitrogen Corporation) was used as a primer for first-strand cDNA synthesis. PCR was performed for 40 cycles of denaturation at 95°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 1 minute with a final 10 minute extension period. The PCR product was electrophoresed on a 1.5% agarose gel, stained with ethidium bromide, and extracted. The 1.6-kb rat PCS cDNA containing the open reading frame sequence (nucleotides 7 to 1512) was cloned in the cytomegalovirus enhancer/promoter-directed vector, which contains a neomycin resistance gene under the control of an SV40 enhancer/early promoter with a polyadenylation signal sequence. A plasmid carrying the lacZ gene substituted for the PCS gene was constructed as a control (pCMV-lacZ). The plasmids were purified with a plasmid purification kit (Qiagen) according to the manufacturer's protocol. The sequence of each plasmid was confirmed by a DNA sequence reader (LI-COR).
Gene Transfer to Cultured Cells With pCMV-PCS
SMCs were explanted from rat aortas and cultured in DMEM
supplemented with 10% FBS (GIBCO), 100 U/mL penicillin, and100 µg/mL
streptomycin. SMCs were grown in 60-mm dishes to 60% confluence and
then transfected with 5 µg pCMV-PCS or pCMV-lacZ with 50 µL of a
liposomal transfection reagent, Lipofectoamin Plus (GIBCO). After
transfection, the SMCs were incubated for 2 days. The medium was
removed once and replaced with 1 mL of fresh medium containing 10
µmol/L sodium arachidonate. At 60 minutes after addition
of arachidonate, the medium was extracted and the
6-keto-PGF1 and TxB2
levels were measured. The concentrations of
6-keto-PGF1 and TxB2
were measured with chemiluminescence immunoassay kits (BioAssay
Inc).
Measurement of PCS Protein Levels
An immunoblotting analysis was performed
using 10% sodium dodecyl sulfate-polyacrylamide gels
with cell lysates.37 The proteins obtained from the SMCs
were transferred to a polyvinyl difluoride membrane (Millipore
Corp.). The membranes were blocked in skim milk, washed 3 times with
Tris-buffered saline with 0.2% Tween-20, and incubated with anti-rat
PCS antibody (1:500 in TBS/Tween-20; Cayman Chemical Co.) for 1 hour at
room temperature. Membranes were washed 3 times with TBS/Tween-20,
incubated with anti-rabbit immunoglobulin antibody (1:5000), washed
again 3 times with TBS/Tween-20, and developed with enhanced
chemiluminescence reagent (Amersham).
Evaluation of Transgene Efficiency in SMCs
Transduced SMCs were detected by LacZ gene
expression using ß-galactosidase staining (5 mmol/L
K4Fe(CN)6, 5 mmol/L
K3Fe(CN)6, 1 mg/mL
5-bromo-4chloro-3-indolyl-b-D-galactpyranoside;
Invitrogen) for 2 hours. Transduction efficiency was evaluated by the
average percentage of positive cells in whole cultured SMCs in 5 to 6
eyefields.
Measurement of cAMP Levels
Cultured SMCs were mixed with 6% trichloroacetate. The solution
was washed with water-saturated diethyl ether 3 times and lyophilized.
cAMP levels at an appropriate dilution were measured by a enzyme
immunoassay (EIA) kit (Amersham) following the manufacturer's
protocol.
PCS Gene Transfer In Vivo
Male Sprague-Dawley rats (Chubu Kagaku Shizai) weighing 300–350
g were maintained under a 12 hour light/12 hour dark cycle at 23°C
and were fed standard laboratory chow (RC4, Oriental Yeast) and water
ad libitum. Rats were anesthetized with 50 mg/kg sodium
pentobarbital, administered intraperitoneally. The
experimental procedure used to induce injury in the rat common carotid
artery has been described in detail elsewhere.8 To prevent
acute thrombosis during the procedure, an intravenous bolus
of heparin (200 U/kg) was injected 5 minutes before vascular injury as
Zoldhelyi et al22 reported. After cervical median
incision, the distal right common artery and the region of bifurcation
was exposed. A 2F balloon catheter (Baxter Healthcare) was inserted
through the external carotid artery and advanced into the thoracic
aorta. After inflation of the balloon, the artery was injured in
position 3 times for 30 seconds each. Both ends of the injured artery
were temporarily ligated, and 30 µg of pCMV-PCS, pCMV-lacZ, or PBS
with 50 µL Lipofectoamin Plus reagent in a total volume of 200 µL
was instilled into the lumen with a 26-gauge needle for 30 minutes. The
catheter was carefully withdrawn and bleeding was prevented with glue
used for thoracic surgery. After confirmation of carotid artery flow
with a flow monitor, the wound was sutured. In a prior study, the
extent of endothelial denudation was confirmed at 2
days after balloon injury by Evans blue staining. Systolic
blood pressure was measured at 7 days by the tail-cuff method (BP98A,
Softron). All experiments were performed in accordance with the
guidelines of the International Committee for Thrombosis and Hemostasis
and were approved by our Ethical Committee for Animal
Experimentation.
PGI2 and TxA2 Production in Rat
Carotid Artery
The carotid arteries treated with pCMV-lacZ and pCMV-PCS were
dissected 7 days after injury (n=6 in each group). The arteries were
cut into 5-mm lengths, washed with PBS, and incubated in 1 mL of
0.1 mmol/L Tris-HCl containing 10 µmol/L sodium
arachidonate at 37°C for 30 minutes. The levels of
6-keto-PGF1 and TxB2 in
the media were measured with the enzyme immunoassay kits described
above.
Localization of Plasmid in the Vessel Wall
Seven days after the instillation of pCMV-PCS and pCMV-lacZ, the
animals were euthanized by the administration of an overdose of
pentobarbital, and the arteries were perfusion-fixed in 2%
glutaraldehyde and 0.2%
paraformaldehyde. LacZ gene expression was
detected by ß-galactosidase staining for 24 hours, and
counterstaining with eosin.
Proliferation Index of SMCs In Vivo
Proliferating SMCs were evaluated by the thymidine analogue BrdU
labeling technique. The proliferation index was measured in the
control, pCMV-lacZ, and pCMV-PCS transfected groups (n=6 in each group)
as Matsuno et al8 reported. BrdU was injected (50 mg/kg,
SC) 1, 8, 16, and 24 hours before removal of the carotid artery at 7
days after vascular injury. BrdU-positive cells were stained with a
murine monoclonal antibody (Amersham), followed by goat anti-mouse Ig
antibodies conjugated to peroxidase and detected with DAB. Adjacent
sections were also stained with hematoxylin for the detection of
nonproliferating cells. The positive and negative nuclei were counted
in the media and newly formed intima. The BrdU labeling index was
calculated by the following formula: (positive nuclei stained by
DAB)/(total nuclei stained by hematoxylin).
Histological Assessment of Neointima
and Vascular Smooth Muscle Layer
Seven or 14 days after injury and transfer, rats were killed and
carotid arteries were perfusion-fixed and harvested for paraffin
embedding. Sections were stained with hematoxylin-eosin or elastica van
Gieson. The extent of neointimal formation was quantified
by computed planimetry of histologically stained
sections. The cross-sectional areas of the blood vessel layers
including the intimal area and medial area were quantified by using NIH
Image (by Wayne Rasband, National Institutes of Health, USA). The
intima/media (I/M) ratios were calculated from 10 to 12
individual cross-sections of each artery. The mean of these
determinations was used to calculate the I/M cross-section ratios for
each animal. For the evaluation of the recovery of
endothelium, sections were incubated overnight with an
anti-von Willebrand factor antiserum conjugated to peroxidase
(1:50; DAKO). After a wash in TBS, the sections were developed with DAB
in Tris-HCl buffer, pH7.6, for 10 minutes, and counterstained with
hematoxylin. The reendothelialization index (reEI) was
defined as the percentage of luminal circumference lined by newly
regenerated endothelium in the inner lumen
circumference. The mean of reEI was calculated from 10 to 12
cross-sections of each artery.
Statistical Analysis
ANOVA followed by the Scheffe's post hoc test was used to
determine significant differences in multiple comparison testing among
the groups. Unpaired Student's t test was used for
comparisons between groups. Fisher's exact probability test was used
for the comparison of the incidence of thrombus occlusion in pCMV-lacZ-
and pCMV-PCS-transfected arteries after balloon injury. All values are
expressed as mean±SEM, and significance was defined as
P<0.05.
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Results |
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Augmentation of Prostacyclin Production in Cultured SMCs by pCMV-PCS Transfer
We initially determined whether the present plasmid vector system could transfer target genes to cultured rat aortic SMCs using pCMV-lacZ. The transgene efficiency in rat aorta SMCs confirmed by ß-galactosidase staining was 21.5±1.7% (n=6). Then, we transfected pCMV-PCS to cultured SMCs to confirm the increment of PGI2 synthesis. The baseline levels of 6-keto-PGF1
produced by pCMV-PCS- and
pCMV-lacZ-transfected SMCs showed no significant difference from those
of untransfected cells (Table
). However,
60 minutes after the 10 µmol/L sodium arachidonate
treatment, the production of
6-keto-PGF1 increased in both pCMV-PCS- and
pCMV-lacZ-transfected SMCs, with a significantly higher
production in the pCMV-PCS-transfected SMCs 2-fold that of the
pCMV-lacZ-transfected cell (P<0.05). In contrast, the
TxB2 production in the SMCs did not
change significantly, even after the treatment with
arachidonic acid.
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PCS protein expression was detected in SMCs by
immunoblotting using a specific monoclonal antibody for
rat PCS. In the pCMV-PCS-transfected cells, 2.1-fold higher PCS protein
expression was observed compared with the pCMV-lacZ transfected SMCs
(n=5 each, P<0.01, Figure 1).
The intracellular cAMP concentration was measured by EIA. The cAMP
level was 2.3 times higher in the pCMV-PCS transfected SMCs compared
with the pCMV-lacZ-transfected cells (17.3±0.5 and 7.5±0.4 pmol/mg
protein, n=5 each, respectively).
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Effects of Gene Transfer on Physiological Parameters
At 7 days after each gene transfer, the rats' systolic
blood pressure was 110±5 mm Hg and 112±7 mm Hg (in the
pCMV-lacZ and pCMV-PCS groups, respectively; n=12 each,
P=NS). Compared with the control values (109±7
mm Hg), no significant difference was observed. Regarding heart rate,
no significant difference was observed among the 3 groups (421±15,
405±12, and 417±14 beats per minute, for pCMV-lacZ, pCMV-PCS, and
control, respectively).
Transduction of the Arterial Vessel Wall After
pCMV-lacZ and pCMV-PCS
The distribution of transgene expression was confirmed 7 days
after lacZ gene transfer in balloon-injured rat carotid
arteries. The pCMV-lacZ-transfected rat carotid arteries showed a
diffuse transduction of the medial layer with a focal transduction of
the adventitia (Figure 2). The
transduction of SMCs in the vessel area amounted to 8.5±0.7% (n=6),
as indicated by the blue coloration of the cell nuclei and cytosol
after pCMV-lacZ transfer. The production of
PGI2 in the arterial vessels was
evaluated by determining the 6-keto-PGF1
levels, 7 days after gene transfer. The
6-keto-PGF1 production levels were
lower in control and pCMV-lacZ-transfected vessels than in uninjured
vessels. In contrast, the level was significantly higher in
pCMV-PCS-transfected vessels than in the control and
pCMV-lacZ-transfected vessels. The PCS gene transfer restored the
6-keto-PGF1 levels to values even higher than
those of the uninjured vessels (n=6 in each group, Figure 3). Thromboxane
A2 (TxA2), the counterpart
of derivatives from PGH2, was also evaluated in
the same samples by determining the levels of thromboxane
B2 (TxB2). There were no
significant differences in the TxB2 levels among
the groups at baseline or after arachidonate treatment.
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Effect of Arterial PCS Gene Transfer on SMC
Proliferation and Neointimal Formation
The inhibitory effect of PGI2 on
SMC proliferation was confirmed by BrdU incorporation rate in the
vessel walls. In the control group, the BrdU incorporation index was
47.6±7.8% (n=5). The PCS gene transfer significantly suppressed the
BrdU incorporation to 7.5±3.3% (n=6, P<0.001 versus
control and pCMV-lacZ), whereas the lacZ gene transfer did
not affect the index (50.7±9.6%, n=6). To investigate whether the
local delivery of the pCMV-PCS gene can accelerate the recovery of
endothelium and reduce neointimal
formation, we conducted a histological analysis
of balloon-injured rat carotid arteries. We first evaluated
reendothelialization after endothelial
denudation. A prominent recovery of endothelium was
observed in the pCMV-PCS-transfected arteries (Figure 4). The mean reEI at 7 days after the
balloon injury was 87.1±6.9% in the pCMV-PCS-transfected arteries
(n=6, P<0.001 versus control and pCMV-lacZ), whereas the
value was 9.0±0.3% and 6.9±0.2% in control and the
pCMV-lacZ-transfected arteries, respectively (n=6 in each group). Even
14 days after the balloon injury, a significant difference was observed
(n=6 in each group, 95.2±8.1%, 61.2±8.7%, and 57.6±7.2% for
pCMV-PCS, control, and pCMV-lacZ, respectively, P<0.01
versus control and pCMV-lacZ). To investigate the effect of gene
transfer on neointimal formation, we calculated the I/M
ratio in each cross-section of artery (Figures 5 and 6).
At 7 days after injury, the I/M ratio was 1.38±0.13 in the control
group and 1.32±0.15 in the pCMV-lacZ group (n=6 in each group,
P=NS). In contrast, the ratio in the pCMV-PCS group was
significantly lower compared with the other two groups (0.16±0.05,
n=6, P<0.001 versus control and pCMV-lacZ). In the
pCMV-PCS-transfected vessels, 88% and 87% reductions of the I/M ratio
were observed compared with the ratio of the control and
pCMV-lacZ-transfected vessels. This effect was also observed at 14
days. The I/M ratios were 1.20±0.14, 1.24±0.15, and 0.14±0.05 for
control, pCMV-lacZ, and pCMV-PCS, respectively (n=6 in each group,
P<0.001 versus control and pCMV-lacZ). In contrast, the
medial layer area was 1.1±0.07 mm2 in the
injured, untransfected arteries and was essentially unchanged after
gene transfer with pCMV-lacZ (1.2±0.08 mm2)
and with pCMV-PCS (1.0±0.03 mm2, n=6 in
each group, P=NS). Intraluminal thrombus formation is often
observed after mechanical stimuli such as balloon injury. No clogging
with thrombus was observed in the pCMV-PCS-transfected vessels, whereas
14% of the vessels were embolized in the pCMV-lacZ-transfected
group (n=30 and 28, respectively, P<0.05).
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Discussion |
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The local delivery of the gene encoding PCS to balloon-injured rat carotid arteries transduced 8.5% of cells in the segment with nonviral lipofection. The overexpression of PCS increased the production of PGI2 in transfected SMCs, and elevated the cAMP levels. We observed abundant regenerated endothelium at the injured site (87.1%) by the single PCS gene transfer and marked reduction of neointimal formation (88%) 7 days after balloon injury. This reduction remained at 14 days after injury.
Many early responses to balloon injury are initiated by platelet
adhesion, aggregation, and thrombus formation followed by the adhesion
and invasion of blood cells such as macrophages and lymphocytes
into arterial walls.31 32 33 34 The interaction of
these blood cells and vessel walls triggers the synthesis and release
of various kinds of growth factors such as platelet-derived growth
factor-, transforming growth factor-ß, and basic fibroblast growth
factor.38 39 These growth factors stimulate SMCs to
migrate from the media into the intima, where they start to proliferate
and secrete extracellular matrix components. The loss of the
endothelial monolayer is associated with the regulatory
dysfunction of these growth factors. Moreover, it is likely that
PGI2 suppresses many of these reactions to
arterial injury. Although each individual protective effect
of PGI2 may not be enough strong, a combination
of such inhibitory effects may occur in multiple steps and
result in marked inhibition of proliferative changes after balloon
injury. This mechanism might be similar to those of other vasodilative
molecules such as c-type natriuretic peptide17
and nitric oxide,20 21 which maintain vascular function
through an autocrine/paracrine loop.
The present results showed that PCS gene transfer accelerated the regrowth of endothelium and restored PGI2 production in injured arteries, events that may lead to the recovery of beneficial functions of endothelium such as the inhibition of thrombus formation and intimal hyperplasia.30 31 32 The mechanism underlying the acceleration of reendothelialization by PGI2 remains unknown. We speculate that through the interaction between PGI2 and some growth factors, such as vascular endothelial growth factor and basic fibroblast growth factor, endothelial cell regrowth may be accelerated.
The vasculoprotective functions of PGI2
including vasodilation, anti-platelet aggregation, anti-leukocyte
adhesion to vessel wall, and SMC proliferation have been known since
the 1970s,24 25 and clinical applications of
PGI2 to proliferative vascular diseases have been
introduced.40 41 42 43 However, the clinical application of
PGI2 has been limited because of its intolerable
systemic, mainly hemorrhagic, side effects.40 41 42 43
Regarding restenosis after angioplasty, Gershlick et
al40 reported that a short-term administration of
PGI2 could not prevent restenosis after
balloon angioplasty. To obtain enough effect, a higher dose of
PGI2 may be required inevitably accompanied by
intolerable side effects. To increase PGI2
production, Zoldhelyi and colleagues22 constructed
a vector carrying the PGHS-1 gene and delivered it to balloon-injured
arteries, resulting in the prevention of thrombus formation after
balloon injury. The overexpression of the PGHS-1 gene also increased
6-keto-PGF1 levels in both cultured cells and
in vessels. However, the alteration of TxA2
catalyzed by the overexpression of the PGHS-1 gene was not evaluated in
their report. An overproduction of PGH2
may induce vascular contraction and platelet aggregation and also
result in an insufficient reduction of neointimal
formation.44 45 46 Pritchard et al47 reported
that after balloon injury, PGH2
production would increase because of the induction of PGHS-2 in
the vessel wall even after a disturbance of constitutive PGHS-1
activity. We speculated that a sufficient supply of
PGH2 may induce an increased production
of PGI2 catalyzed by overexpressed PCS in damaged
vessel walls. In support of our speculation, Shitashige et
al48 investigated the mechanism of the different
utilizations of arachidonic acid between PGHS-1 and
PGHS-2 and reported that a small amount (<2.5 µmol/L) of
arachidonic acid released by some stimuli is converted
exclusively by PGHS-2. In the present study, the
6-keto-PGF1 levels were elevated in the
arteries transfected with PCS gene without an increase in the
TxA2 level, suggesting that the overexpression of
PCS may increase PGI2 production without
affecting TxA2 synthesis after balloon injury. In
addition, we did not observe hemorrhagic side effects locally or
systemically in the PCS-gene transfected rats; this may be related to
the imbalance of PGI2 and
TxA2 production.49
In the present study, we transfected the rat PCS gene into carotid arteries with a nonviral lipofection method, resulting in prolonged inhibition of neointimal formation. As a methodology of gene transfer, lipofection is inferior to virus-mediated transfection in expression efficiency (4% to 5% and 10% to 30%, respectively).12 13 14 15 16 17 18 19 20 21 22 23 50 However, virus-mediated gene transfection has several limitations. For example, adenovirus vectors retain most the parent virus genome, which is associated with undesired gene expression that results in both immune and vascular inflammatory responses.50 Other virus-mediated vectors such as adeno-associated virus and retrovirus have recently been developed to reduce these undesirable responses, but they have limitations regarding the convenience of preparation or with repeated usage.33 34 50 We confirmed that the liposomal reagent used in the present study had enough potential transgene efficiency (8.5% in transfected vessel cells) and markedly inhibited restenosis after arterial injury. In transgene studies, it is well known that the bystander effect is involved in the inhibition of vascular SMC proliferation.51 Our results show that as few as 8.5% of cells transduced with PCS gene affected the entire cells of the carotid artery vasoprotectively (ie, accelerated reendothelialization and inhibition of neointimal formation) through this bystander effect.
Several problems must be resolved before the clinical application of gene therapy using PCS gene transfer to prevent restenosis. We focused mainly on neointimal hyperplasia at the balloon-injured site. In humans, however, it is well known that elastic recoil (within one day after angioplasty) and shrinkage at the late phase (about 3 to 6 months) may be involved in restenosis.2 3 4 52 53 54 In this respect, further studies are required before adding gene therapy as an alternative therapeutic strategy for restenosis.
In conclusion, the local delivery of PCS gene markedly inhibited neointimal formation after balloon injury in rat carotid artery with accelerated reendothelialization. PCS gene transfer may be a novel gene therapeutic strategy for restenosis after angioplasty.
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Acknowledgments |
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This study was performed mainly at the Institute for Laboratory Animal Research, Nagoya University.
Received July 31, 1998; accepted October 2, 1998.
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