(Circulation. 1996;93:10-17.)
© 1996 American Heart Association, Inc.
Articles |
From The University of TexasHouston 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 TexasHouston Medical School, 6431 Fannin, MSB 5.016, Houston, TX 77030.
| Abstract |
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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 |
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| Methods |
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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-1treated 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 cDNAtransduced 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 |
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contents in the medium were
measured by RIA. The results are shown in Table 1
produced by Ad-COX-1
and Ad-LacZtransfected cells were not significantly different from
those of untransfected cells (Table 1
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.
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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-1infected 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.
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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-1infected 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).
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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
.
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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-LacZinfected 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
).
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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 |
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, 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-catheterinjured 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 controltreated 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 |
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| Acknowledgments |
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| Footnotes |
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| Appendix |
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Received October 16, 1995; accepted November 5, 1995.
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