(Circulation. 1996;93:10-17.)
© 1996 American Heart Association, Inc.
Articles |
Prevention of Arterial Thrombosis by Adenovirus-Mediated Transfer of Cyclooxygenase Gene
From The University of Texas–Houston Health Science Center (P.Z., J.M., X.-M.X., D.L.-M., L.M.B., J.T.W., K.K.W.); University of Texas Southwestern Medical School, Dallas (R.S.M.); and Texas Heart Institute, Houston, Tex (F.J.C., L.M.B., J.T.W.).
Correspondence to Kenneth K. Wu, MD, Professor and Director, Division of Hematology, and Vascular Biology Research Center, Department of Medicine, University of Texas–Houston Medical School, 6431 Fannin, MSB 5.016, Houston, TX 77030.
 |
Abstract |
|---|
 Top Abstract IntroductionMethods Results Discussion Appendix References |
|---|
Background Prostacyclin is an important vasoprotective molecule. It inhibits platelet aggregation, monocyte interaction with endothelium, and smooth muscle cell lipid accumulation. Vascular cyclooxygenase-1 (COX-1) is the rate-limiting step in prostacyclin synthesis. The objective of this study was to determine whether adenovirus-mediated transfer of COX-1 could restore COX-1 activity, augment prostacyclin synthesis, and prevent thrombus formation in a porcine carotid angioplasty model.
Methods and Results Human COX-1 cDNA driven by a
cytomegalovirus promoter was constructed into a
replication-defective adenovirus 5 vector by homologous
recombination. Recombinant adenovirus without a foreign gene (Ad-RR)
and buffer were included as controls. Recombinant Ad-LacZ was used for
marking the transfected cells in vivo. In the in vitro experiments,
cultured human endothelial cells (ECs) and porcine
arterial smooth muscle cells (SMCs) were incubated with
Ad-COX-1 for 2 hours and 6-keto-PGF1
level and the
transgene expression were determined 72 hours after infection. In the
in vivo experiments, recombinant adenoviruses were directly instilled
into angioplasty-injured porcine carotid arteries for 30 minutes.
Cyclic flow changes were monitored for 10 days and thrombus formation
was examined histologically thereafter. Transgene
expression and prostaglandin I2
(PGI2) synthesis by the injured arteries were determined.
Cultured ECs infected with Ad-COX-1 produced a fivefold to eightfold
increase in PGI2, and the transgene expression in
cultured porcine SMCs was demonstrated by Northern analysis.
Direct administration of Ad-COX-1 at a dose of 3x1010 pfu
completely inhibited carotid cyclic flow changes and thrombus formation
accompanied by a fourfold increase in PGI2 synthesis by the
injured arteries 10 days after infection, whereas Ad-COX-1 at a lower
dose, 5x109 pfu, had no antithrombotic effects when
compared with Ad-RR vector and buffer controls.
Conclusions Adenovirus-mediated transfer of COX-1 to angioplasty-injured carotid arteries was efficacious in augmenting PGI2 synthesis and was associated with an inhibition of thrombosis when a relatively high titer of adenovirus was instilled.
Key Words: thrombosis • prostaglandins • gene transfer • angioplasty
|
Introduction |
|---|
|
TopAbstractIntroductionMethods Results Discussion Appendix References |
|---|
Prostacyclin is a key vasoprotective molecule that is synthesized in vascular cells and acts as an autacoid in inhibiting platelet aggregation and maintaining SMC relaxation.1 2 3 Its biosynthesis is catalyzed in succession by phospholipase A2, which liberates arachidonic acid (AA) from the sn-2 position of membrane phospholipids; cyclooxygenase (also known as prostaglandin H synthase), which converts the liberated AA into PGG2 and then PGH2; and finally prostacyclin synthase, which transforms PGH2 into prostacyclin.4 Injury to the endothelium may cause a reduction in prostacyclin synthesis with an attendant risk of thrombosis. Attempts to treat thrombosis by systemic prostacyclin infusion have been unsatisfactory because of unacceptable complications.5 6 Experimental data have indicated that the constitutive COX-1 or PGH synthase-1 is the key rate-limiting step in controlling the extent of prostacyclin synthesis.7 8 9 Overexpression of COX-1 in a cultured human endothelial cell line by retrovirus-mediated transfer of COX-1 cDNA is accompanied by a sustained increase in prostacyclin synthesis.10 The in vitro gene transfer results suggest that local production of prostacyclin to the vascular injury sites may be augmented by transfer of COX-1. However, retroviral vectors are inefficient for direct gene transfer to the damaged vessel wall.11 By contrast, replication-defective recombinant adenoviruses are efficient vectors for direct in vivo arterial gene transfer.12 In the present study, we constructed a replication-defective recombinant Ad5 viral vector containing a human COX-1 cDNA (Ad-COX-1) insert driven by cytomegalovirus promoter and showed its ability to augment COX-1 expression and PGI2 synthesis in cultured vascular cells and injured porcine carotid arteries. Direct instillation of Ad-COX-1 into angioplasty-injured porcine carotid arteries significantly reduced CF changes and was associated with the inhibition of carotid arterial thrombosis. The antithrombotic effect is achieved only at a relatively high titer of Ad-COX-1.
|
Methods |
|---|
|
TopAbstractIntroductionMethods Results Discussion Appendix References |
|---|
Preparation of Recombinant Adenovirus
Construction and Preparation of Replication-Defective Recombinant Adenovirus
Ad5/CMV-COX-1 (Ad-COX-1), Ad5/CMV-LacZ (Ad-LacZ), and Ad5 vector control (Ad-RR) were essentially as previously reported.13 14 High-titer stocks of recombinant viruses were prepared by a modification of the method described previously.14 Confluent monolayers of transformed human embryonic kidney (293) cells were infected by exposure to primary recombinant viruses for 1 hour at a multiplicity of infection of 0.01 and incubated in Dulbecco's modified Eagle's medium containing 4.5 g/L glucose supplemented with 2% fetal bovine serum until extensive cytopathic effects were observed. Three to 5 days after infection, 293 cells were lysed in isotonic saline buffer (mmol/L: Tris HCl 10, NaCl 137, KCl 4, MgCl2 1, pH 7.4) and 0.1% Nonidet P-40. The lysate was centrifuged at 12 000g, and virus was precipitated from the supernatant by addition of 0.5 vol of 20% polyethylene glycol/2.5 mol/L NaCl. The precipitated virus was resuspended and purified by cesium chloride density centrifugation and sepharose CL4B chromatography. The cells were stored at -80°C in phosphate-buffered saline containing 0.1 mg/mL bovine serum albumin until use. Titers of purified virus preparations were determined by plaque assay on monolayers of 293 cells. Virus preparations were not screened for the presence of wild-type virus, which had not been reported at the time virus was grown for the experiments reported here.
Infection of Cultured Vascular Cells With Ad-COX-1
Cultured
human ECs, EA.hy 926,15 or porcine aortic
SMCs16 were incubated with fresh medium containing
Ad-COX-1 (60 pfu/cell) or vehicle for 2 hours. The cells were washed
and incubated in fresh medium for 48 hours. Prostacyclin content in the
medium produced by the infected, cultured ECs was measured as
6-keto-PGF1 by a highly sensitive RIA.17
The mRNA expression in the infected porcine SMCs was determined by
Northern blot analysis by using a 1.3-kb human COX-1
riboprobe,17 which is highly selective for COX-1
mRNA.7 To control for differences in the efficiency of
total RNA extraction or RNA transfer to the membranes, porcine
glyceraldehyde 3-phosphate dehydrogenase probed with an
[-32P]-labeled human cDNA was included as an
internal
control.
Porcine Carotid Crush-Injury and Angioplasty Model
Because of
its simplicity, a standardized, crush-injury
model18 was initially used to establish whether
enhancement of PGI2 synthesis by adenovirus-mediated
transfer of COX-1 cDNA in vivo was feasible. Because a single carotid
cutdown is used to allow for the creation of crush injury and virus
delivery, surgery is rapid and relatively low cost, not requiring
x-ray and angioplasty equipment.
After the feasibility of enhancing PGI2 synthesis by COX-1 cDNA transfer was established, an animal model bearing similarity to events taking place during angioplasty in humans was used to study the effects of enhanced PGI2 synthesis on thrombus formation. Pig carotid arteries were injured by an angioplasty procedure modified from that of Steele et al.19 In addition to complete endothelial denudation in all balloon-damaged arteries, deep arterial injury (tear extending beyond the internal elastic membrane) is observed in 50% of damaged arteries in this model.19 In preliminary experiments, a constrictor was applied within the first hour of angioplasty to the center of the injury, often leading to irreversible thrombotic occlusion despite administration of heparin to ACTs of 500 to 800 seconds. However, by 2 hours from injury, cyclic thrombus deposition in the injured artery spontaneously decreased and a constrictor could be applied without promoting irreversible occlusion in the majority of vessels. By 12 hours from injury, only rare cyclic flow variations were observed. To prevent acute thrombosis during surgery in the study, an intravenous bolus of heparin (200 U/kg body wt) was given 5 minutes before angioplasty; this prolonged the ACT to threefold to fivefold higher than the basal ACT. ACT prolongation was comparable in all experimental groups. Bilateral carotid arteries were exposed and 5 mL 1% lidocaine was applied. A 5F-polyethylene balloon catheter was introduced into either common carotid artery via the right femoral artery, and once the catheter was in place under fluoroscopy, the balloon was inflated for 30 seconds x5 with a 60-second interval between each inflation. Both ends of the injured carotid segments were temporarily ligated, and Ad-COX-1, Ad-RR, or buffer was instilled into the lumen with a 22-gauge Teflon catheter for 30 minutes. Five minutes before removing the instillate and allowing reflow, we administered a second bolus of heparin (100 U/kg). Carotid flow was monitored for 2 hours before application of a constrictor, and thereafter the pigs were allowed to recover. Heparin 50 U · kg-1 · h-1 was given for the first 24 hours to all pigs.
Carotid flow was continuously recorded for 10 days according to a procedure previously described.20 Despite a high degree of heparin anticoagulant activity evidenced by prolonged ACT, severe CF reductions were noted during the first 2 hours in all animals, with frequent zero-flow requiring massage of the artery to dislodge thrombi. The flow became stabilized thereafter. There were no differences in the initial flow reductions among various experimental groups of animals. The flow rate at 24 hours after surgery was used as the postangioplasty baseline for assessment of carotid flow changes. Heparin was discontinued after the initial 24 hours. A "zero" flow was defined as a complete stop of flow for at least 24 hours. When zero-flow was present at the time of euthanasia (one Doppler reading was taken at this time in all pigs), the presence of an occlusive thrombus was confirmed by gross and histological examination in 100% of cases. All the animals that developed zero-flow had persistent zero-flow for the entire period of study except for one control animal in which the flow returned on day 9. This was thought to be due to spontaneous fibrinolysis. The severity of CF variations is arbitrarily classified according to the extent of CF reductions. A reduction of CF >75% was considered severe; 25% to 74%, moderate; and <25%, mild. The frequency of CF changes during the entire monitoring period was compared between Ad-COX-1 and the two control groups. At the end of 10 days, 10 000 U heparin were administered prior to euthanasia with a barbiturate overdose. Animal experiments were approved by the Animal Care Committee.
Histological Examinations
Common carotid arteries were
removed en bloc and postfixed in
10% buffered formalin for 
72 hours. Sections were paraffin embedded
by using standard laboratory procedures and stained with
hematoxylin-eosin or Movat's pentachrome stain. Paraffin
sections were evaluated by light microscopy as to the extent of
thrombosis and neointimal proliferation. The morphological
evaluation was performed in a blinded fashion. For
ß-galactosidase staining, Ad-LacZ (6x109 pfu/mL) was
instilled into the angioplasty-injured artery according to
identical procedures as described above. The animals were killed on day
10, and the carotid arterial tissues were prepared and
stained with X-Gal by procedures previously
reported.21
Prostacyclin Formation in Porcine Carotid
Arterial Segments
The injured porcine carotid arterial segments
treated with Ad-COX-1, Ad-RR, or buffer were dissected on day 10 after
the animals were killed. The arterial segments were cut
into rings of approximately equal size. The arterial rings
were gently washed and incubated with fresh PBS, pH 7.4, containing 20
µmol/L arachidonic acid at 37°C for 30 minutes. The
medium was removed and the content of the stable metabolite of
prostacyclin, 6-keto-PGF1, was measured by a
specific and sensitive RIA.
Study Groups
The effect of Ad-COX-1, Ad-RR, and mock saline
on carotid blood
flow and thrombus deposition after injury was studied in 48 animals. In
addition, 3 animals received recombinant adenovirus carrying the LacZ
gene (Ad-LacZ) to monitor the duration of gene expression independent
of PGI2 expression. Isolated, balloon-injured carotid
segments were incubated for 30 minutes with 3.2x1010 pfu
of Ad-RR, 5x109 pfu of Ad-COX-1, 3x1010 pfu
of Ad-COX-1, or buffer alone. Each viral dose was suspended in about
0.5 to 0.7 mL buffer. This volume was found to completely fill the
isolated arterial segments under an instillation pressure
of 300 to 500 mm Hg. Two animals assigned to Ad-COX-1 and one control
pig were excluded from analysis because of irreversible
thrombotic occlusion within the first 6 hours after surgery. In
addition, one control and one Ad-COX-1–treated pig tore off the wires
connected to the probes on day 7 and 8, respectively, and were excluded
from analysis because of lack of an interpretable flow signal
and the presence of uncontrolled, added trauma to the artery. CFVs and
histological thrombus at death were analyzed in
8 pigs receiving 3x1010 pfu of Ad-COX, 17 receiving
5x109 pfu Ad-COX-1, 5 receiving 3.2x1010 pfu
Ad-RR, and 13 pigs treated with buffer as additional control.
Whole-blood aggregation was performed in a Chronolog Lumi-aggregometer (model 560VS) in all pigs. Aggregation was stimulated with type I collagen (2, 5, and 10 µg/mL), the thromboxane A2 analog U46619 (0.5, 1, and 2 µg/mL), thrombin (2.5 and 5 U/mL), and ADP (0.05, 0.1, and 0.2 µmol/L). Except for blood used for aggregation with thrombin and ADP (collected in 1/9 vol of 3.8% citrate), blood was drawn into heparin (final concentration 4 U per milliliter of blood). Platelet-rich plasma was prepared for aggregation with ADP. Platelet counts in control pigs were 324 800±110 366 at baseline and 275 800±143 000 at 10 days. The relative platelet counts were 384 666±95 980 and 363 670±115 590 in COX-1 cDNA–transduced pigs. Hematocrits (baseline and 10-day values) were 28.98±3.36 and 29.68±2.44 in control pigs and 25.02±2.89 and 28.48±2.47 in gene-treated pigs.
Statistical Analysis
ANOVA was used to determine the presence
of significant
(P<.05) differences in end points between the study groups.
Multiple-comparison testing with the Student-Newman-Keuls test was
then carried out to isolate the groups producing significant
differences.
|
Results |
|---|
|
TopAbstractIntroductionMethods Results Discussion Appendix References |
|---|
Augmentation of Prostacyclin Production in Cultured Human Endothelial Cells by Ad-COX-1 Infection
We initially determined whether Ad-COX-1 infection of cultured EA hy 926 human EC line increased PGI2 synthesis. EA hy 926 is a hybrid cell line that possesses morphological and biochemical characteristics of a human EC.15 We have previously shown that retrovirus-mediated transfer of COX-1 into this cell line leads to a sustained overexpression of COX-1.10 Monolayers of confluent EA cells in T-25 flasks were treated with Ad-COX-1 (60 pfu/cell) or Ad-LacZ at 37°C for 2 hours. For comparison, EA cells were incubated in cultured medium alone at 37°C for 2 hours. The cells were washed and stimulated with arachidonate (10 µmol/L), ionophore A23187 (10 µmol/L), or buffer alone at 37°C for 10 minutes. 6-Keto-PGF1
contents in the medium were
measured by RIA. The results are shown in Table 1
. The
baseline levels of 6-keto-PGF1 produced by Ad-COX-1–
and Ad-LacZ–transfected cells were not significantly different from
those of untransfected cells (Table 1). Arachidonate and
ionophore treatments of Ad-COX-1 cells were accompanied by about a
fivefold and eightfold increase in 6-keto-PGF1 levels,
respectively. By contrast, the 6-keto-PGF1 levels
produced by Ad-LacZ cells in response to arachidonate or
ionophore were not different from those of untransfected native cells.
These results indicate overexpression of COX-1 activity in Ad-COX-1
cells. This effect is not due to a nonspecific adenoviral effect on
COX-2 induction, as the Ad-LacZ transfected cells produced essentially
identical baseline and stimulated levels of 6-keto-PGF1
as the native untransfected cells.
|
Expression of COX-1 Transgene in Cultured Porcine
SMCs
After denudation by angioplasty, the SMCs are the main cell type
to be exposed to the recombinant adenoviruses. To determine whether
vascular SMCs took up the recombinant Ad-COX-1, cultured porcine
arterial SMCs were treated with Ad-COX-1 (60 pfu/cell) for
2 hours. COX-1 mRNA was determined by Northern blot analysis,
using a 1.3-kb riboprobe for COX-1. As shown in Fig 1,
uninfected cells exhibited a single 2.7-kb COX-1 band, whereas in
addition to this inherent COX-1 mRNA band, the Ad-COX-1–infected SMCs
expressed a 3.2-kb band, suggestive of transcription of the 430-bp SV40
polyadenylation sequence present in the adenoviral vector
downstream from the COX-1 cDNA.
|
Effect of Ad-COX-1 Transfer on Thrombus Formation
In
preliminary experiments, we used a simple porcine carotid
injury model to determine whether instillation of Ad-COX-1 for 30
minutes would elicit an increased PGI2 synthesis. Porcine
carotid arteries were injured by modified Spencer-Welles forceps
according to a procedure described by Butler et al.18
Ad-COX-1 (1x1010 pfu suspended in 0.5 mL buffer [titer
2x1010 pfu/mL]) or buffer alone was instilled in the
injured arterial segments for 30 minutes before to reflow.
The animals were euthanitized at 72 hours after infection, and carotid
arteries were isolated and dissected into rings. PGI2
synthesis by Ad-COX-1–infected arteries was threefold higher than that
of buffer controls.
The effect of direct adenovirus-mediated transfer
of COX-1 on
thrombus formation was evaluated in a porcine carotid angioplasty
model.19 Carotid arterial CFVs that occurred
during the 10-day period and thrombus formation on the damaged
arterial wall at the end of the 10-day period were compared
between pigs treated with 5x109 pfu and
3x1010 pfu Ad-COX-1, pigs treated with buffer alone, and
pigs treated with an adenoviral vector without foreign genes (Ad-RR,
3.2x1010 pfu). Five pigs were excluded from
analysis (see "Methods"). The incidence of CFVs and
histological thrombus at death in the 43 pigs is
presented in Table 2. All pigs developing
zero-flow during the time of flow monitoring had occlusive carotid
thrombi at death. Neither the ACTs during the first 24 hours nor the
response of platelets to agonists at baseline and 10 days,
including the thromboxane A2 analogue U46619
and thrombin, were significantly different between groups (data not
shown).
|
Without exception, pigs that had received
5x109 pfu
Ad-COX-1 developed frequent CFVs, which was not statistically different
from the buffer or the Ad-RR control groups. In contrast, no CF
reduction or thrombus deposition was found in the eight pigs that had
been treated with 3x1010 pfu Ad-COX-1, which retained
normal flow throughout the study period from day 2 to 10, whereas only
one buffer control pig retained normal flow, and 54% of the buffer
control and 40% of the Ad-RR control animals developed zero-flow
(Table 2
). Thirty percent of pigs receiving
5x109 pfu of
Ad-COX-1 developed zero-flow. A representative set
of tracings comparing CFVs between a buffer control and a
high-titer Ad-COX-1 pig is shown in Fig 2.
|
Histological
examinations of the injured carotid
arteries at 10 days after infection confirmed the presence of occlusive
thrombi in animals exhibiting zero-flows (Fig 3).
Despite a similar extent of vascular injury comparable to control and
low-titer Ad-COX-1 groups, thrombi were not detected in any of the
eight pigs infected with 3x1010 pfu of Ad-COX-1 (Fig
3).
Expression of ß-galactosidase in Ad-LacZ–infected carotid
arteries was evaluated by staining the arterial wall in
situ with X-Gal. Positive staining was noted in small numbers of
vascular cells scattered throughout the injured arterial
segment 10 days after Ad-LacZ infection (Fig 3).
|
Prostacyclin Synthesis in Arterial Segments Infected
With Recombinant Adenovirus
The level of prostacyclin production by
angioplasty-injured arterial rings on day 10 was
fourfold to fivefold higher in pigs receiving 3x1010 pfu
Ad-COX-1 (137±74 ng/mL) than in those receiving the buffer
(32±4.8
ng/mL) or Ad-RR (24±2.7 ng/mL) control. The prostacyclin levels
produced in the group infected with 5x109 pfu Ad-COX-1
(35±5 ng/ml) were not different from those of the control groups.
|
Discussion |
|---|
|
TopAbstractIntroductionMethods Results Discussion Appendix References |
|---|
It has recently been shown in the retrovirus-mediated COX-1 overexpressed human endothelial cell line that, despite a short COX-1 half-life of about 10 minutes due to COX-1 autoinactivation during catalysis, not all the enzymes are autoinactivated; only about 30% of the overexpressed COX-1 is inactivated during each catalysis.7 PGI2 accounts for the overwhelming majority of cyclooxygenase products generated by endothelial and vascular SMCs, 95% and 73%, respectively.22 Most of the rest is accounted for by PGE2, PGF2
, and
PGD2, while thromboxane A2
is produced in only very small amounts by endothelium
and vascular SMCs.22 In agreement with these studies, we
found that after retrovirus-mediated transfer of COX-1 into
endothelial cells, overexpression of PGI2
accounted for most of the prostanoids elaborated by
endothelial cells.7 Thus, overexpression
of COX-1 results in a sustained production of a high level of
PGI2. These findings lend credence to COX-1 gene transfer.
As replication-defective retroviral vectors are inefficient for
direct arterial gene transfer, we tested the efficacy of
replication-defective Ad-COX-1 transfer in cultured cells and a
porcine angioplasty model. Infection of cultured ECs with Ad-COX-1 for
a short time period of 2 hours leads to a severalfold increase in
PGI2 synthesis at 72 hours after infection. Transgene
expression is also evident in porcine SMCs under a similar experimental
protocol. Furthermore, direct instillation of Ad-COX-1 into a carotid
arterial segment injured with mechanical clamps for 30
minutes increased PGI2 production by about
threefold. These results are consistent with several recent
reports on direct arterial gene transfer by adenoviral
vectors and support the notion that adenoviral vectors are feasible for
transferring COX-1 cDNA into damaged arterial wall for
augmenting PGI2 synthesis. Our results indicate that Ad-COX-1 transfer into the angioplasty-injured porcine carotid arterial wall immediately after injury is effective in protecting the injured arteries from developing CF changes and thrombus formation. Thrombus deposition immediately after injury was prevented by administration of high-dose heparin (total of 300 U/kg). In addition, application of the constrictor to the injured artery was delayed by 2 hours after completion of angioplasty and incubation of the injured arteries with virus or control buffer. Preliminary experiments indicated that earlier addition of stenosis to the injury caused irreversible occlusion in the majority of animals. Between 2 and 12 hours after injury, spontaneous thrombus formation in this model gradually abates. Only 3 of 45 pigs developed irreversible carotid occlusion between 2 and 72 hours after angioplasty. In a similar injury model in the dog, the early nadir in the number of CFVs was observed to coincide with degranulation of platelets and their nearly complete refractoriness to several agonists, including the thromboxane analog U46619 (Willerson JT, 1995, unpublished observations). Recurrent thrombus deposition, mirrored by CFVs, returned about 3 days after angioplasty in Ad-RR– and saline-treated carotid arteries. Clearly, the antithrombotic effects of Ad-COX-1 gene transfer in our study did not protect against thrombus deposition immediately after balloon injury, which required high-dose heparin for its prevention. On the other hand, chronic recurrent thrombus deposition plays an important role in the acute and chronic progression of atherothrombotic disease,23 24 25 and the ability to continuously monitor arterial flow after injury has revealed the important role played by recurrent thrombus deposition in the development of chronic stenosis after experimental injury.20 CFVs also were observed in humans after angioplasty and were abolished by platelet antagonists.26 27 In our model, CFVs tended to fade spontaneously by day 10, in accordance with the reported time course of endothelial healing.19 Thus, the duration of CFVs in this model coincides with that of foreign gene expression by recombinant first-generation adenoviruses, making these vectors suitable for testing of antithrombotic gene therapy.
The antithrombotic effect was conferred by a relatively high viral dose of Ad-COX-1 (3x1010 pfu). Lowering the dose to 5x109 pfu resulted in a disappearance of the protective properties against CF changes and thrombus formation. PGI2 synthesis by carotid arteries in the high-titer Ad-COX-1 group was increased over the control groups by fourfold at 10 days after infection, whereas PGI2 synthesis in the low-titer Ad-COX-1 group was not different from controls. Although thromboxane A2 levels were not measured in this study, these findings suggest that the platelet agonist thromboxane A2 was not a major product after COX-1 gene transfer to vascular cells or injured arteries in vivo.
Induction of COX-2 is unlikely to explain our results in vitro and in vivo. First, infection of porcine SMCs in vitro resulted in a 3.2-kb message hybridizing with the COX-1 riboprobe. We speculate, but do not prove, that the 3.2-kb band represents additional transcription of the 430-bp SV40 polyadenylation sequence present in the adenoviral vector downstream from the 2.7-kb COX-1 cDNA. Since there is about 60% homology between the COX-1 and COX-2 messages, we would have expected a hybridization of the riboprobe to the 4.3-kb COX-2 message, had COX-2 been induced.28 No 4.3-kb message was detected in the SMCs. Second, although the inducible COX-2 has been shown to be expressed in balloon-catheter–injured arteries and may contribute to the control level of PGI2 synthesis,29 this level of PGI2 synthesis appears to be inadequate for protecting against thrombosis. As the transgene expression in adenovirus-mediated transfer tends to dissipate 2 to 3 weeks after infection,12 PGI2 production may be much higher during the immediate periods after infection than those on day 10. Sustained elevations of PGI2 production in the high-titer Ad-COX-1 group during the 10-day period are likely to be responsible for the antithrombotic effects. Third, no elevation in PGI2 synthesis in arterial rings stimulated ex vivo or thrombus protection in vivo was noted after infection of the artery with Ad-RR at a dose identical to that of Ad-COX-1.
Taken together, these data suggest that direct adenovirus-mediated transfer of a physiologically relevant gene, COX-1, at a sufficiently high titer is capable of restoring COX-1 expression and PGI2 synthesis and is associated with preventing angioplasty-induced arterial thrombosis in a pig model. Beneficial effects on pulmonary arterial pressure have been shown by liposome-mediated transfer of COX-1.30 If our observations are confirmed in similar and other experimental models in the future, gene transfer of COX-1 may be considered as a possible, future therapeutic modality for human arterial thrombotic disorders. However, there are a number of problems that must be solved before COX-1 gene therapy may be applied to human diseases. For example, adenovirus-mediated gene transfer may be less efficient when applied to atherosclerotic arteries.31 In most human arterial thrombotic diseases, thrombus is formed on atherosclerotic lesions. Hence, a high titer of adenoviruses or other more efficient vectors may be required for transferring the COX-1 gene to achieve an efficacious antithrombotic effect. Unfortunately, adenoviruses at high titers are associated with inflammation due to immune reactions, which have been most prominent after adenoviral gene transfer to liver14 32 and lung.33 We were unable to show that Ad-COX-1 at 6x1010 pfu/mL (a dose of 3x1010 pfu) caused inflammation beyond that associated with the angioplasty in our model by day 10. Only scant, mostly adventitial, mononuclear infiltrates were observed in both virus- and saline control–treated arteries, possibly related to the severe medial balloon injury or external manipulation of the artery during surgery. In agreement with earlier reports,34 35 delivery of recombinant adenovirus to the artery at the doses used in this study seems to be associated with minimal or no inflammation.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This work was supported by NIH grants HL-35387, HL-50179, HL-17669, and NS-23327; American Heart Association Grant-in-Aid 92008550; and Texas Advanced Technology program 00366020. We thank C.J. Edgell for providing EA.hy926 cells.
|
Footnotes |
|---|
Guest editor for this article was Valentin Fuster, MD, Mt Sinai Medical Center, New York, NY.
|
Appendix |
|---|
|
TopAbstractIntroductionMethods Results Discussion Appendix References |
|---|
Effects of Ad-LacZ
Although one batch of high-titer Ad-LacZ, later found to be contaminated with endotoxin, had shown protection against thrombosis, not associated with an increase in PGI2 synthesis, subsequent experiments with a second, endotoxin-free batch of Ad-LacZ at the original titer (6x1010 pfu/mL) and at a 50% higher and a 50% lower titer (9x1010 and 3x1010 pfu/mL), respectively, failed to show vasoprotection. In these experiments, Ad-LacZ treatment was associated with CFV and thrombus incidence not different from treatment with Ad-RR and buffer control. Further, in vitro experiments with Ad-LacZ failed to show an increase in PGI2 synthesis or expression of inducible nitric oxide synthase. The issue of "vasoprotection" seen with the endotoxin-contaminated batch of high-titer Ad-LacZ initially used was not pursued further.
Received October 16, 1995; accepted November 5, 1995.
|
References |
|---|
|
TopAbstractIntroductionMethods Results Discussion Appendix References |
|---|
1. Moncada S, Gryglewski RJ, Bunting S, Vane JR. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature. 1976;263:663-665. [Medline] [Order article via Infotrieve]
2.
Vane JR. Prostaglandins and the
cardiovascular system. Br Heart J. 1983;49:405-409.
3.
Pomerantz K, Hajjar D. Eicosanoids in
regulation of arterial smooth muscle cell
phenotype, proliferative capacity, and cholesterol
metabolism.
Arteriosclerosis. 1989;9:413-429.
4. Wu KK, Kulmacz RJ, Wang L-H, Loose-Mitchell DS, Tsai A-L. Molecular biology of prostacyclin biosynthesis. In: Rubanyi G, Vane JR, eds. Prostacyclin: New Perspectives for Basic Research and Novel Therapeutic Indications. Amsterdam, Netherlands: Elsevier Science Publishers BV; 1992:11-23.
5. Szczeklik A, Nizankowski R, Skawinski S, Szczeklik J, Gluszko P, Gryglewski RJ. Successful therapy of advanced atherosclerosis obliterans with prostacyclin. Lancet. 1979;1:1111-1114. [Medline] [Order article via Infotrieve]
6. Belch JJF, Newman P, Drury JK, McKenzie F, Capell H, Leiberman P, Forbes CD, Prentice CRM. Intermittent prostacyclin infusion in patients with Raynaud's syndrome. Lancet. 1983;1:313-315. [Medline] [Order article via Infotrieve]
7.
Sanduja SK, Tsai A-L, Matijevic-Aleksic N, Wu KK.
Kinetics of prostacyclin synthesis in a PGHS-1 overexpressed
endothelial cell. Am J Physiol. 1994;267:C1459-C1466.
8. Kent RS, Diedrich SL, Whorton R. Regulation of vascular prostaglandin synthesis by metabolites of arachidonic acid in perfused rabbit aorta. J Clin Invest. 1983;72:455-465.
9. McIntire TM, Zimmerman GA, Satoh K, Prescott SM. Cultured endothelial cells synthesize both PAF and PGI2 in response to histamine, bradykinin and ATP. J Clin Invest. 1985;76:271-280.
10. Xu X-M, Ohashi K, Sanduja SK, Ruan K-H, Wang L-H, Wu KK. Enhanced prostacyclin synthesis in endothelial cells by retrovirus-mediated transfer of prostaglandin H synthase cDNA. J Clin Invest. 1993;91:1843-1849.
11.
Flugelman MY, Jaklitsch MT, Newman KD, Casscells SW,
Brattauer GL, Dichek DA. Low level in vivo gene transfer into
the arterial wall through a perforated balloon
catheter. Circulation. 1992;85:1110-1117.
12.
Lee SW, Trapnell BC, Rade JJ, Virmani R, Dichek DA.
In vivo adenoviral vector–mediated gene transfer into
balloon-injured rat carotid arteries. Circ
Res. 1993;73:797-807.
13.
Gomez-Foix AM, Coats WS, Baque S, Alam T, Gerard RD,
Newgard CB. Adenovirus-mediated transfer of the muscle
glycogen phosphorylase gene into hepatocytes
confers altered regulation of glycogen metabolism.
J Biol Chem. 1992;267:25129-25134.
14. Herz J, Gerard RD. Adenovirus-mediated transfer of low density lipoprotein receptor gene acutely accelerates cholesterol clearance in normal mice. Proc Natl Acad Sci U S A. 1993;980:2812-2816.
15.
Edgell CJ, McDonald CC, Graham JB. Permanent
cell line expressing human factor VIII-related antigen established by
hybridization. Proc Natl Acad Sci U S A. 1983;80:3734-3738.
16.
Ross R. The smooth muscle cell, II: growth of
smooth muscle in culture and formation of elastic fibers.
J Cell Biol. 1971;50:172-186.
17.
Wu KK, Sanduja R, Ferhanoglu B, Loose-Mitchell DS.
Aspirin inhibits interleukin 1-induced prostaglandin
H synthase expression in cultured endothelial
cells. Proc Natl Acad Sci U S A. 1991;88:2384-2387.
18. Butler KD, Ambler J, Dolan S, Giddings J, Talbot MD, Wallis RB. A non-occlusive model of arterial thrombus formation in the rat and its modification by inhibitors of platelet function, or thrombin activity. Blood Coagul Fibrinolysis. 1992;3:155-165. [Medline] [Order article via Infotrieve]
19.
Steele PM, Chesebro JH, Stanson AW, Holmes DR, Dewanjee
MK, Badimon K, Fuster V. Balloon angioplasty: natural history of
the pathophysiological response to injury in a
pig model. Circ Res. 1985;57:105-112.
20.
Willerson JT, Yao GK, McNatt J, Benedict CR, Anderson
HV, Golino P, Murphree SS, Buja LM. Frequency and severity of
cyclic flow alternations and platelet aggregation predict the
severity of neointimal proliferation following experimental
coronary stenosis and endothelial
injury. Proc Natl Acad Sci U S A. 1991;88:10624-10628.
21.
Willard JE, Landau C, Glamann DB, Burns D, Jessen ME,
Pirwitz MJ, Gerard RD, Meidell RS. Genetic modification
of the vessel wall: comparison of surgical and catheter-based
techniques for delivery of recombinant adenovirus.
Circulation. 1994;89:2190-2197.
22. Coughlin SR, Moskowitz MA, Zetter BR, Antoniades HN, Levine L. Platelet-dependent stimulation of prostacyclin synthesis by platelet-derived growth factor. Nature. 1980;288:600-602. [Medline] [Order article via Infotrieve]
23.
Chen L, Chester MR, Redwood S, Huang J, Leatham E,
Kaski JC. Angiographic stenosis progression and
coronary events in patients with `stabilized unstable
angina.' Circulation. 1995;91:2319-2324.
24.
Duguid JB. Mural thrombosis in arteries.
Br Med Bull. 1955;11:36-38.
25.
Falk E. Unstable angina with fatal outcome:
dynamic coronary thrombosis leading to infarction and/or sudden
death. Circulation. 1985;71:699-708.
26. Eichhorn EJ, Grayburn PA, Willard JE, Anderson HV, Bedotto JB, Carry M, Kahn JK, Willerson JT. Spontaneous alterations in coronary blood flow velocity before and after coronary angioplasty in patients with severe angina. J Am Coll Cardiol. 1991;17:43-52. [Abstract]
27. Anderson HV, Kirkeeide RL, Krishnaswami A, Weigelt LA, Revana M, Weisman HF, Willerson JT. Cyclic flow variations after coronary angioplasty in humans: clinical and angiographic characteristics and elimination with 7E3 monoclonal antiplatelet antibody. J Am Coll Cardiol. 1994;23:1031-1037. [Abstract]
28.
Jones DA, Carlton DP, McIntyre TM, Zimmerman GA,
Prescott SM. Molecular cloning of human
prostaglandin endoperoxide synthase type II
and demonstration of expression in response to
cytokines. J Biol Chem. 1993;268:9049-9054.
29.
Pritchard KA Jr, O'Banion MK, Miano JM, Vlasic N,
Bhatia UG, Young DA, Stemerman MB. Induction of
cyclooxygenase-2 in rat vascular smooth muscle
cells in vitro and in vivo. J Biol Chem. 1994;269:8504-8509.
30. Conary JT, Parker RE, Christman BW, Faulks RD, King GA, Meyrick BO, Brigham KL. Protection of rabbit lungs from endotoxin injury by in vivo hyperexpression of the prostaglandin G/H synthase gene. J Clin Invest. 1994;93:1834-1840.
31. Feldman LJ, Steg PG, Zheng LP, Chen D, Kearney M, McGarr SE, Barry JJ, Dedieu J-F, Perricaudet M, Isner JM. Low-efficiency of percutaneous adenovirus-mediated arterial gene transfer in the atherosclerotic rabbit. J Clin Invest. 1995;95:2662-2671.
32.
Engelhardt JF, Ye X, Doranz B, Wilson JM.
Ablation of E2A in recombinant adenoviruses improves transgene
persistence and decreases inflammatory response in mouse liver.
Proc Natl Acad Sci U S A. 1994;91:6196-6200.
33. Bout A, Perricaudet M, Baskin G, Imver JL, Scholte BJ, Pavirani A, Valerio D. Lung gene therapy: in vivo adenovirus-mediated gene transfer to rhesus monkey airway epithelium. Hum Gene Ther. 1994;5:3-10. [Medline] [Order article via Infotrieve]
34.
Lemarchand P, Jones M, Yamada I, Crystal RG. In
vivo gene transfer and expression in normal uninjured blood vessels
using replication-deficient recombinant adenovirus vectors.
Circ Res. 1993;72:1132-1138.
35.
Schulick AH, Newman KD, Virmani R, Dichek DA. In
vivo gene transfer into injured carotid arteries: optimization and
evaluation of acute toxicity. Circulation. 1995;91:2407-2414.
This article has been cited by other articles:
 |
|
 |
|
  K. Yokouchi, Y. Numaguchi, R. Kubota, M. Ishii, H. Imai, R. Murakami, Y. Ogawa, T. Kondo, K. Okumura, D. E. Ingber, et al. l-Caldesmon Regulates Proliferation and Migration of Vascular Smooth Muscle Cells and Inhibits Neointimal Formation After Angioplasty Arterioscler Thromb Vasc Biol, October 1, 2006; 26(10): 2231 - 2237. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. H. Tan, S.-A. Xue, M. Manunta, S. C. Beutelspacher, H. Fazekasova, A.K.M. Shamsul Alam, M. O. McClure, and A. J.T. George Effect of Vectors on Human Endothelial Cell Signal Transduction: Implications for Cardiovascular Gene Therapy Arterioscler Thromb Vasc Biol, March 1, 2006; 26(3): 462 - 467. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T.-N. Lin, W.-M. Cheung, J.-S. Wu, J.-J. Chen, H. Lin, J.-J. Chen, J.-Y. Liou, S.-K. Shyue, and K. K. Wu 15d-Prostaglandin J2 Protects Brain From Ischemia-Reperfusion Injury Arterioscler Thromb Vasc Biol, March 1, 2006; 26(3): 481 - 487. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Li, C. Zhang, S. Schaefer, A. Estes, and K. U. Malik ANG II-induced neointimal growth is mediated via cPLA2- and PLD2-activated Akt in balloon-injured rat carotid artery Am J Physiol Heart Circ Physiol, December 1, 2005; 289(6): H2592 - H2601. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-C. Shyu, S.-Z. Lin, M.-F. Chiang, D.-C. Ding, K.-W. Li, S.-F. Chen, H.-I Yang, and H. Li Overexpression of PrPC by Adenovirus-Mediated Gene Targeting Reduces Ischemic Injury in a Stroke Rat Model J. Neurosci., September 28, 2005; 25(39): 8967 - 8977. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Liu, Z.-Q. Chen, G. C. Bobustuc, J. M. McNatt, H. Segall, S. Pan, J. T. Willerson, and P. Zoldhelyi Local Gene Transduction of Cyclooxygenase-1 Increases Blood Flow in Injured Atherosclerotic Rabbit Arteries Circulation, April 12, 2005; 111(14): 1833 - 1840. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Sharif, K. Daly, J. Crowley, and T. O'Brien Current status of catheter- and stent-based gene therapy Cardiovasc Res, November 1, 2004; 64(2): 208 - 216. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Wen, S. Graf, P. G. Massey, and D. A. Dichek Improved Vascular Gene Transfer With a Helper-Dependent Adenoviral Vector Circulation, September 14, 2004; 110(11): 1484 - 1491. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Tulis, Z. H. Mnjoyan, R. L. Schiesser, H. S. Shelat, A. J. Evans, P. Zoldhelyi, and K. Fujise Adenoviral Gene Transfer of Fortilin Attenuates Neointima Formation Through Suppression of Vascular Smooth Muscle Cell Proliferation and Migration Circulation, January 7, 2003; 107(1): 98 - 105. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-Y. Qian, A. Haruno, Y. Asada, T. Nishida, Y. Saito, T. Matsuda, and H. Ueno Local Expression of C-Type Natriuretic Peptide Suppresses Inflammation, Eliminates Shear Stress-Induced Thrombosis, and Prevents Neointima Formation Through Enhanced Nitric Oxide Production in Rabbit Injured Carotid Arteries Circ. Res., November 29, 2002; 91(11): 1063 - 1069. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Connolly, D. J. Bouchier-Hayes, E. Kaye, A. Leahy, D. Fitzgerald, and O. Belton Cyclooxygenase Isozyme Expression and Intimal Hyperplasia in a Rat Model of Balloon Angioplasty J. Pharmacol. Exp. Ther., February 1, 2002; 300(2): 393 - 398. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B.S. Wung, J.J. Cheng, S.-K. Shyue, and D.L. Wang NO Modulates Monocyte Chemotactic Protein-1 Expression in Endothelial Cells Under Cyclic Strain Arterioscler Thromb Vasc Biol, December 1, 2001; 21(12): 1941 - 1947. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. T. Davidge Prostaglandin H Synthase and Vascular Function Circ. Res., October 12, 2001; 89(8): 650 - 660. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Golino, P. Cirillo, P. Calabro', M. Ragni, D. D'Andrea, E. V. Avvedimento, F. Vigorito, N. Corcione, F. Loffredo, and M. Chiariello Expression of exogenous tissue factor pathway inhibitor in vivo suppresses thrombus formation in injured rabbit carotid arteries J. Am. Coll. Cardiol., August 1, 2001; 38(2): 569 - 576. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-K. Shyue, M.-J. Tsai, J.-Y. Liou, J. T. Willerson, and K. K. Wu Selective Augmentation of Prostacyclin Production by Combined Prostacyclin Synthase and Cyclooxygenase-1 Gene Transfer Circulation, April 24, 2001; 103(16): 2090 - 2095. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Zoldhelyi, Z.-Q. Chen, H. S. Shelat, J. M. McNatt, and J. T. Willerson Local gene transfer of tissue factor pathway inhibitor regulates intimal hyperplasia in atherosclerotic arteries PNAS, March 27, 2001; 98(7): 4078 - 4083. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. G. Priori, C. Napolitano, N. Tiso, M. Memmi, G. Vignati, R. Bloise, V. Sorrentino, and G. A. Danieli Mutations in the Cardiac Ryanodine Receptor Gene (hRyR2) Underlie Catecholaminergic Polymorphic Ventricular Tachycardia Circulation, December 6, 2000; (2000) 11. [Abstract] [Full Text]
|
|
|
|
|
|
J. M. Waugh, J. Li-Hawkins, E. Yuksel, M. D. Kuo, P. N. Cifra, P. R. Hilfiker, R. Geske, M. Chawla, J. Thomas, S. M. Shenaq, et al. Thrombomodulin Overexpression to Limit Neointima Formation Circulation, July 18, 2000; 102(3): 332 - 337. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-Y. Liou, S.-K. Shyue, M.-J. Tsai, C.-L. Chung, K.-Y. Chu, and K. K. Wu Colocalization of Prostacyclin Synthase with Prostaglandin H Synthase-1 (PGHS-1) but Not Phorbol Ester-induced PGHS-2 in Cultured Endothelial Cells J. Biol. Chem., May 12, 2000; 275(20): 15314 - 15320. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. P. Rentrop Thrombi in Acute Coronary Syndromes : Revisited and Revised Circulation, April 4, 2000; 101(13): 1619 - 1626. [Full Text] [PDF]
|
|
|
|
|
|
P. Zoldhelyi, J. McNatt, H. S. Shelat, Y. Yamamoto, Z.-Q. Chen, and J. T. Willerson Thromboresistance of Balloon-Injured Porcine Carotid Arteries After Local Gene Transfer of Human Tissue Factor Pathway Inhibitor Circulation, January 25, 2000; 101(3): 289 - 295. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Vinals, J. Martinez-Gonzalez, and L. Badimon Regulatory Effects of HDL on Smooth Muscle Cell Prostacyclin Release Arterioscler Thromb Vasc Biol, October 1, 1999; 19(10): 2405 - 2411. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Harada, Y. Toki, Y. Numaguchi, H. Osanai, T. Ito, K. Okumura, and T. Hayakawa Prostacyclin synthase gene transfer inhibits neointimal formation in rat balloon-injured arteries without bleeding complications Cardiovasc Res, August 1, 1999; 43(2): 481 - 491. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nishida, H. Ueno, N. Atsuchi, R. Kawano, Y. Asada, Y. Nakahara, Y.-i. Kamikubo, A. Takeshita, and H. Yasui Adenovirus-Mediated Local Expression of Human Tissue Factor Pathway Inhibitor Eliminates Shear Stress–Induced Recurrent Thrombosis in the Injured Carotid Artery of the Rabbit Circ. Res., June 25, 1999; 84(12): 1446 - 1452. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Massberg, M. Sausbier, P. Klatt, M. Bauer, A. Pfeifer, W. Siess, R. Fassler, P. Ruth, F. Krombach, and F. Hofmann Increased Adhesion and Aggregation of Platelets Lacking Cyclic Guanosine 3',5'-Monophosphate Kinase I J. Exp. Med., April 19, 1999; 189(8): 1255 - 1264. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Numaguchi, K. Naruse, M. Harada, H. Osanai, S. Mokuno, K. Murase, H. Matsui, Y. Toki, T. Ito, K. Okumura, et al. Prostacyclin Synthase Gene Transfer Accelerates Reendothelialization and Inhibits Neointimal Formation in Rat Carotid Arteries After Balloon Injury Arterioscler Thromb Vasc Biol, March 1, 1999; 19(3): 727 - 733. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. J. Kullo, R. D. Simari, and R. S. Schwartz Vascular Gene Transfer : From Bench to Bedside Arterioscler Thromb Vasc Biol, February 1, 1999; 19(2): 196 - 207. [Full Text] [PDF]
|
|
|
|
 |
|
 T. Todaka, C. Yokoyama, H. Yanamoto, N. Hashimoto, I. Nagata, T. Tsukahara, S. Hara, T. Hatae, R. Morishita, M. Aoki, et al. Gene Transfer of Human Prostacyclin Synthase Prevents Neointimal Formation After Carotid Balloon Injury in Rats • Editorial Comment Stroke, February 1, 1999; 30(2): 419 - 426. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Cheng, G. Mantile, R. Pauly, C. Nater, A. Felici, R. Monticone, C. Bilato, Y. A. Gluzband, M. T. Crow, W. Stetler-Stevenson, et al. Adenovirus-Mediated Gene Transfer of the Human Tissue Inhibitor of Metalloproteinase-2 Blocks Vascular Smooth Muscle Cell Invasiveness In Vitro and Modulates Neointimal Development In Vivo Circulation, November 17, 1998; 98(20): 2195 - 2201. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Baek and K. L. March Gene Therapy for Restenosis : Getting Nearer the Heart of the Matter Circ. Res., February 23, 1998; 82(3): 295 - 305. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. H Baker, D. Mehta, S. J George, and G. D Angelini Prevention of vein graft failure: potential applications for gene therapy Cardiovasc Res, September 1, 1997; 35(3): 442 - 450. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. D Gerard and D. Collen Adenovirus gene therapy for hypercholesterolemia, thrombosis and restenosis Cardiovasc Res, September 1, 1997; 35(3): 451 - 458. [Full Text] [PDF]
|
|
|
|
|
|
G. Vassalli and D. A Dichek Gene therapy for arterial thrombosis Cardiovasc Res, September 1, 1997; 35(3): 459 - 469. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Singh, S. Pan, C. S. Mueske, T. Witt, L. S. Kleppe, T. E. Peterson, A. Slobodova, J.-Y. Chang, N. M. Caplice, and R. D. Simari Role for Tissue Factor Pathway in Murine Model of Vascular Remodeling Circ. Res., July 6, 2001; 89(1): 71 - 76. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. E. Burleigh, V. R. Babaev, J. A. Oates, R. C. Harris, S. Gautam, D. Riendeau, L. J. Marnett, J. D. Morrow, S. Fazio, and M. F. Linton Cyclooxygenase-2 Promotes Early Atherosclerotic Lesion Formation in LDL Receptor-Deficient Mice Circulation, April 16, 2002; 105(15): 1816 - 1823. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Lin, T.-N. Lin, W.-M. Cheung, G.-M. Nian, P.-H. Tseng, S.-F. Chen, J.-J. Chen, S.-K. Shyue, J.-Y. Liou, C.-W. Wu, et al. Cyclooxygenase-1 and Bicistronic Cyclooxygenase-1/Prostacyclin Synthase Gene Transfer Protect Against Ischemic Cerebral Infarction Circulation, April 23, 2002; 105(16): 1962 - 1969. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||