(Circulation. 2001;103:570.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Combination of a Brief Irrigation With Tissue Factor Pathway Inhibitor (TFPI) and Adenovirus-Mediated Local TFPI Gene Transfer Additively Reduces Neointima Formation in Balloon-Injured Rabbit Carotid Arteries
From the Departments of Cardiology (N.A., A.T., H.U.) and Cardiovascular Surgery (T.N.), Graduate School of Medical Sciences, Kyushu University, Fukuoka; the Department of Pathology, Miyazaki Medical College, Miyazaki (K.M., Y.A.); and Chemo-Sero-Therapeutic Research, Kumamoto (Y.K.), Japan. Dr Ueno is now at the Department of Biochemistry and Molecular Pathophysiology, University of Occupational and Environmental Health, Kitakyushu, Japan.
Correspondence to Hikaru Ueno, MD, PhD, Professor, Department of Biochemistry and Molecular Pathophysiology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu, 807-8555 Japan. E-mail hueno{at}med.uoeh-u.ac.jp
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Abstract |
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Background—Tissue factor pathway inhibitor (TFPI) is a physiological antagonist of TF. We tested whether a brief irrigation with TFPI protein (rTFPI) or TFPI gene transfer into injured arteries would suppress TF activity and reduce fibroproliferative changes and investigated whether a combination of these methods would show an additive effect.
Methods and Results—We prepared adenoviruses expressing either TFPI (AdTFPI) or bacterial ß-galactosidase (AdLacZ). Rabbit carotid arteries were balloon-injured and either infected with AdTFPI (or AdLacZ) or irrigated briefly with rTFPI (or saline). After injury, TF activity in arteries increased and was sustained; however, it was suppressed during the initial 24 hours by rTFPI irrigation (but not by gene transfer) and for a substantial period of time by TFPI gene transfer (but not by rTFPI irrigation). Four weeks later, the ratio of the intimal to medial areas was 34.3±8.7% (mean±SD, n=14) in saline-treated arteries and 33.3±4.2% in AdLacZ-infected arteries (P=NS versus saline). However, it was reduced to 25.5±8.5% in rTFPI-irrigated arteries (P<0.01 versus saline) and to 20.7±5.3% in AdTFPI-infected arteries (P<0.01 versus AdLacZ). With a combination of irrigation and gene transfer, the ratio was further reduced to 12.6±4.7% (P<0.01 versus rTFPI, P<0.05 versus AdTFPI). Systemic coagulation status was not affected in these animals.
Conclusions—A combination of rTFPI irrigation and TFPI gene transfer overcomes the shortcomings shown by each method when used alone and achieves a full coverage of TF activity suppression, thereby enhancing their therapeutic effects without systemic side effects. This combination may be an effective strategy for the prevention of thrombosis and proliferative changes after angioplasty in humans.
Key Words: anticoagulants • gene therapy • platelets • thrombin • thrombus
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Introduction |
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Tissue factor (TF), a 47-kDa transmembrane glycoprotein, binds and activates factor VII, which then activates factors IX and X, thus initiating the blood coagulation pathway,1 2 leading to generation of thrombin. Thrombin is an inducer of fibrin formation and also a potent mitogen for the cells of the arterial wall.3 In normal arteries, TF is expressed only in the adventitia.4 In atherosclerotic arteries, however, it is expressed ubiquitously in the plaques.5 6 Furthermore, once injury occurs, TF becomes expressed within hours in almost all layers of the arterial wall.7 8 Accumulating evidence suggests that TF may have a variety of actions in addition to initiating thrombin formation. For example, TF VIIa induces the activation of mitogen-activated protein kinase9 and evokes intracellular Ca2+ mobilization,10 which may lead to cell proliferation. The finding that TF expression in the neointima is sustained for 8 weeks after injury11 and a report that thrombin activity in arteries is detectable for 2 weeks after balloon injury12 both suggest that TF and thrombin may be involved in the proliferative response after arterial injury and that anti-TF treatment could be effective not only in inhibiting thrombus formation but also in suppressing neointima formation.
Intact endothelial cells produce an anti-TF molecule, known as tissue factor pathway inhibitor (TFPI). TFPI, a Kunitz-type protease inhibitor, directly inhibits the factor Xa and the factor VIIa/TF catalytic complex.13 14 Because TF is an initiator of the coagulation cascade, we expected that TFPI would be a more effective molecule against thrombin than a direct thrombin antagonist such as hirudin.
It has been shown repeatedly that adenovirus-mediated gene transfer into arteries can evoke site-specific production of recombinant protein for a prolonged period of time.15 16 17 In fact, we recently observed that adenovirus-mediated local expression of TFPI eliminates shear-stress–induced recurrent thrombosis in injured rabbit carotid arteries for 2 weeks, even in the presence of catecholamine, without inducing any apparent systemic side effects.18 One inherent limitation of gene therapy in any acute setting, such as vascular injury during angioplasty, however, is the lag time for adequate expression of the vector-encoded protein. An irrigation with recombinant TFPI protein (rTFPI) may be able to cover this lag phase, and a combination of rTFPI and gene transfer may show an additive therapeutic effect. The aim of this study was to test this hypothesis.
We first investigated whether local delivery of TFPI, either by a single brief irrigation with rTFPI or by adenovirus-mediated gene transfer, would suppress TF activation and neointima formation in balloon-injured arteries. Furthermore, we examined whether a combination of these 2 methods could achieve a full coverage of TF activity suppression and show any enhanced inhibitory effect on neointima formation.
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Methods |
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Preparation of Adenoviruses
Replication-defective E1- and E3- adenoviral vectors expressing human TFPI (AdTFPI)18 or bacterial ß-galactosidase (AdLacZ)19 20 under a CA promoter21 were prepared as previously described.18 22
In Vivo Gene Transfer Into Injured
Arteries
All animals were treated under protocols approved by
the animal care committee of Kyushu University. The experiment was
carried out in accordance with both the Guidelines for Animal
Experiments in Kyushu University and the Law (No. 105) and Notification
(No. 6) of the Japanese government. Balloon injury and in vivo gene
transfer into carotid arteries of Japanese White rabbits (male,
weighing 3200±180 g) were performed as previously
described.18 After
heparinization (1500 U/kg), the isolated space within injured arteries
was filled with saline (
0.2 mL), AdTFPI, or AdLacZ
(7.5x108 pfu/mL) for 20 minutes. Then,
arteries were irrigated with either rTFPI (300 µg) or saline for 20
minutes. The titer of adenovirus used in this study was below the
inflammatory threshold (1.6x109 pfu/mL), as
recently reported.23 We did
not notice any significant or consistent histological differences
between arteries subjected to balloon injury and arteries given an
injury followed by adenoviral infection, as previously
reported.18 19
After various treatments, some segments were harvested, then placed in DMEM with 10% serum for 24 hours at 37°C. The amount of TFPI protein in the medium was measured by a 1-step sandwich ELISA method with human recombinant TFPI as a standard, as described previously.18 24 The minimum amount of TFPI detectable by this method is 6.3 ng/mL.
The vessels were harvested 4 weeks later, and 4 sections (5 µm thick) were cut from the middle portion of each vessel. The cross-sectional areas of neointima and media were measured morphometrically with an automated computer-based image analyzer (DKC-5000, SONY) by a technician blinded to the treatment regimen. The ratio of intimal to medial area (I/M ratio) was calculated. The mean value from 4 sections was counted as the 1 value for the rabbit. Fourteen rabbits for each group were analyzed.
In Vivo TF Activity
Segments of injured arteries (10 mm long) were
harvested, and the adventitia was carefully stripped off. TF activity
was measured and expressed in arbitrary units, as previously
described.11 The protein
concentration was determined with Bradford reagent
(Bio-Rad).
Immunohistochemistry
Some carotid arteries were perfusion-fixed with 4%
paraformaldehyde and embedded in paraffin, then sections were examined
immunohistochemically with the standard
streptavidin-biotin-immunoperoxidase staining method (Histofine SAB-PO
kit, Nichirei). Monoclonal antibodies against either human TFPI
(American Diagnostica Inc) or the Ki-67 antigen (Dako) were used.
Preimmune serum was used as a negative control.
Electron Microscopic Analysis
Seven days after the gene transfer, shear stress was
applied by constriction for 30 minutes as previously
reported,18 then arteries
(n=4 in each group) were resected and subjected to a scanning electron
microscopic analysis as previously
described.25 A central area
of injury (10 mm long) in each artery was examined (S-800
microscope, Hitachi).
Statistical Analysis
One-way ANOVA followed by the post hoc test was used
to determine significant differences in multiple comparison among
groups. A level of P<0.05 was
considered statistically
significant.
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Results |
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TFPI Protein in rTFPI-Irrigated and AdTFPI-Infected Arteries
After TFPI gene transfer, TFPI protein was immunohistostained in a broad area of the arterial wall, whereas no staining was observed in the AdLacZ-infected artery (Figure 1
). No signal was detectable in the AdTFPI-treated
artery if preimmune serum was used instead of the primary antibody (not
shown). At that time, we did not know why TFPI protein was detectable
in a wide area, even though it is a secreted protein.
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To examine how long TFPI protein remained or when it was
generated after rTFPI irrigation or TFPI gene transfer, respectively,
treated arteries were resected 1 hour, 24 hours, or 3 or 25 days after
injury and treatments and incubated ex vivo in culture medium for 24
hours. Then, TFPI antigen in the medium was measured by human
TFPI-specific ELISA. A considerable amount of TFPI was still detectable
in the medium from the rTFPI-irrigated artery resected 24 hours after
irrigation, although no TFPI was detectable when harvested 3 days after
irrigation
(Table 1). From the AdTFPI-infected arteries, only a
moderate amount of TFPI was generated during 1 to 2 days after
infection, but the amount increased by 5-fold in arteries resected 3
days after infection. Notably, TFPI production was still detectable 25
days after gene transfer.
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TF Activity in Arteries Is Suppressed by rTFPI
Irrigation and TFPI Gene Transfer
To examine whether the TFPI present or produced in
arteries after irrigation or gene transfer, respectively, possesses
biological activity, we measured TF activity in the arteries. After
injury, TF activity increased from 0.79±0.18 AU/mg protein (mean±SD,
n=6) to 1.12±0.26 AU/mg protein at 2 hours and to 1.67±0.30 AU/mg
protein at 4 hours (n=6,
P<0.01 versus control). The
increased activity of TF remained for 
7 days; it was 1.62±0.74 AU/mg
protein (n=6) at 24 hours and 1.55±0.35 AU/mg protein (n=4) on day 7
after injury. Interestingly, a single brief irrigation with rTFPI
significantly reduced the TF activity at 4 hours (0.98±0.28 AU/mg
protein) and 24 hours (0.96±0.1 AU/mg protein) after injury
(Figure 2). The TF activity in the AdTFPI-infected arteries
was not significantly reduced (1.2±0.38, n=6) at 24 hours; however, it
was significantly reduced on day 7 (0.65±0.05 AU/mg protein, n=6) to a
level even lower than that seen in intact arteries. These results
indicate that in the 24 hours after injury, TF activation was largely
suppressed after a single irrigation with rTFPI and that AdTFPI
infection led to a sustained suppression of the TF activity. The data
indicate that a combination of these 2 methods could be effective in
inhibiting TF activity for a prolonged period of time starting
immediately after injury.
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Absence of Fibrin Formation and Platelet
Aggregation in the AdTFPI-Infected Arteries
Seven days after injury and gene transfer, arteries
under shear stress were subjected to an electron microscopic analysis.
The luminal surface of the AdLacZ-infected artery was covered with a
large number of aggregated platelets in which fibrin and many
erythrocytes were entrapped
(Figure 3, right). In contrast, in the AdTFPI-infected
artery, only a monolayer of spread-out platelets was seen
(Figure 3, left), with neither platelet aggregation nor
fibrin formation being observed.
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Single Brief Irrigation With rTFPI and Local
TFPI Gene Transfer Each Suppresses Neointima Formation in
Balloon-Injured Arteries
Next, we examined whether local application of TFPI,
either by irrigation with rTFPI or by TFPI gene transfer, can suppress
proliferative changes in injured arteries. Balloon-injured arteries
were first dwelled with saline, then irrigated with either
saline or rTFPI. Some injured arteries were infected with either AdLacZ
or AdTFPI, then irrigated with saline. Four weeks later, neointima
formation was examined histologically. The medial area did not vary
significantly among the arteries examined
(Figure 4A). A single rTFPI irrigation significantly reduced
neointima formation (I/M ratio 25.5±8.5%, mean±SD, n=14,
P<0.05) compared with that
seen in saline-irrigated arteries (34.3±8.7%, n=14)
(Figure 4B). AdTFPI-infected arteries also showed a
significant inhibition (20.7±5.3%, n=14,
P<0.05 versus AdLacZ) compared
with that seen in AdLacZ-infected arteries (33.3±4.2%, n=14)
(Figure 4B). There were no significant differences between
the saline-infused and saline-irrigated arteries and the
AdLacZ-infected and saline-irrigated arteries.
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Cell proliferation activity in arteries 3 days after injury and gene transfer was semiquantified by immunohistostaining with a monoclonal antibody to the Ki-67 antigen, which detects proliferating cells.26 The Ki-67 labeling index (the proportion of all nuclei that stained positively with Ki-67) was calculated as a percentage for medial smooth muscle cells. Although many Ki-67–positive smooth muscle cell nuclei were observed in the media of saline-treated (Ki-67 labeling index was 43.1±8.1%, mean±SD, 4 fields from each of 4 rabbits, total 16 fields for each group) and AdLacZ-infected (39.3±7.4%, P=NS versus saline) arteries, significantly low numbers of nuclei were positively stained in the media of both rTFPI-treated (22.8±6.8%, P<0.05 versus saline) and AdTFPI-infected (7.8±6.5%, P<0.01 versus AdLacZ [and saline]) arteries.
Combination of rTFPI Irrigation and TFPI Gene
Transfer Additively Reduces Neointima Formation
Finally, we examined whether an enhanced reduction in
neointima formation could be achieved by a combination strategy (a
single irrigation with rTFPI plus TFPI gene transfer). This strategy
produced the greatest reduction (I/M ratio 12.6±4.7%, n=14) among the
arteries tested in this study
(Figure 4B
). The reduction was additive and significantly
greater than those achieved either by rTFPI irrigation alone (preceded
by saline incubation) (25.5±8.5%,
P<0.05) or by TFPI gene
transfer alone (followed by saline irrigation) (20.7±5.3%,
P<0.05).
No Significant Changes in PT and aPTT in Plasma
of rTFPI-Irrigated and AdTFPI-Infected Rabbits
Prothrombin time (PT) and activated partial
thromboplastin time (aPTT) in rabbits were measured with an Amelung
coagulometer, as previously
described.18 Neither PT nor
aPTT was altered in AdTFPI-infected rabbits 7 days after gene transfer
(Table 2), in which gene expression might be
submaximal.27 Immediately
after irrigation with rTFPI (300 µg), PT and aPTT became slightly
longer than before treatment; however, the difference was not
statistically significant. With heparin, PT and aPTT were greatly
prolonged. In the presence of heparin, no significant further
prolongation of PT and aPTT was observed after a subsequent irrigation
with rTFPI
(Table 2).
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Discussion |
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It takes at least a few hours before a transferred gene becomes expressed as a protein, and it may take much more time for the protein to reach sufficient concentration to show biological effects (see Table 1
). An irrigation with rTFPI should cover this lag
phase until sufficient TFPI protein can be generated long-term by TFPI
gene transfer. The aim of this study was to test this hypothesis. In
fact, TF activity was increased and sustained after injury, but it was
significantly suppressed by rTFPI irrigation in the early phase (the
initial 24 hours) and by AdTFPI infection in the late phase
(Figure 2). Each method led to significant reduction in
neointima formation
(Figure 4) without affecting the systemic coagulation status
(Table 2). These results suggest that 2 periods of TF
activation (and probably thrombin, fibrin, and thrombus formation, see
Figure 3) (ie, in the early and late phases) may contribute
independently to the fibroproliferative changes in injured arteries.
Most importantly, we demonstrated that a combination of these 2 methods
achieved an additive reduction in neointima formation
(Figure 4). This is probably through a suppression of TF
activity for a prolonged period of time starting immediately after
injury
(Figure 2). To the best of our knowledge, this study is the
first solid evidence showing the usefulness of a combination of
recombinant protein and gene transfer, which overcame the shortcomings
of each method by enhancing their therapeutic effects and avoiding
systemic side effects.
It is still controversial whether mural thrombosis persists
for a long time after balloon injury. Some workers have reported that
no thrombus can be found 48 hours after
injury.28 29
Conversely, Gallo et al12
showed that thrombin activity in the plasma is detectable even 2 weeks
after such injury. Hatakeyama et
al6 reported that the level
of TF mRNA and its activity increased continuously in the neointima for
2 to 8 weeks after injury. In our study, an enhanced activity of TF
persisted for 1 week after injury
(Figure 2). Together, these findings suggest that TF
activation and thrombin generation may be sustained for a prolonged
period of time after injury. Although it is difficult to evaluate in a
quantitative manner whether or not thrombus and fibrin formation
actually occurred, our electron microscopic examination revealed that
they were present in the AdLacZ-infected arteries, whereas only
platelet adherence was seen in the AdTFPI-treated arteries, even under
shear stress
(Figure 3). TF and thrombin seem to have many biological
effects in addition to initiating thrombosis, and thus, the sustained
activation and/or generation of these molecules might be expected to be
involved in fibroproliferative responses. Our finding that TFPI
treatment leads to a reduction in neointima formation is consistent
with this notion. However, the possibility that TFPI may have some
direct inhibitory effects on cell proliferation and migration cannot be
excluded.
An interesting point in this study is that a substantial
amount of TFPI indeed remained in the arteries after a single brief
irrigation with rTFPI (300 µg for 20 minutes)
(Table 1) and that this irrigation with rTFPI achieved a
significant suppression of TF activity, at least in the initial 24
hours
(Figure 2), and a significant reduction in neointima
formation
(Figure 4). Oltrona et
al30 reported that only a
continuous infusion for 24 hours of a relatively large amount of TFPI
(0.5 mg/kg bolus plus 100
µg · kg-1 · min-1
for 24 hours), but not an infusion for 3 hours, reduced neointima
formation in balloon-injured pig carotid arteries. We do not know the
explanation for this discrepancy. The method used for the
administration (local irrigation versus intravenous infusion) and/or
species differences (rabbits versus pigs) may be relevant factors.
Other antithrombosis reagents, such as hirudin, also require a
continuous infusion for as long as several
days.12 Interestingly, it
was reported that TFPI remained at the surface of the injured arterial
wall for 3 days, although its activity was not
examined.31 In this study,
we also observed that a substantial amount of rTFPI indeed remained in
arteries even 24 hours after irrigation, and furthermore, we confirmed
that it had a biological effect. The detailed mechanism underlying this
sustained survival of rTFPI needs to be clarified in future studies.
TFPI inhibits an initial step in the coagulation pathway; thus, it
should efficiently inhibit new thrombin formation. These features of
TFPI (long survival and being an inhibitor at the initial step of the
coagulation pathway) should be beneficial and of practical advantage in
clinical practice. It must be admitted that the reduction in neointima
formation after adenovirus-mediated TFPI gene transfer into rabbit
carotid arteries (the present study) was similar to that after
adenovirus-mediated hirudin gene transfer into rat carotid
arteries.32 However, direct
comparison in the same species needs to be done to determine which of
these molecules is the more effective at suppressing thrombosis and
proliferative changes in injured arteries.
In summary, our study demonstrated that a combination of rTFPI irrigation and TFPI gene transfer can suppress TF activation for a prolonged period of time starting immediately after injury and achieve an additive reduction in neointima formation without inducing systemic bleeding. This combined therapy may prove to be an effective and relatively safe strategy for the prevention of thrombosis and restenosis after angioplasty in humans.
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Acknowledgments |
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This study was supported by grants from the Ministry of Education, Science, and Culture of Japan; from the Ryoichi Naito Foundation for Medical Research, Osaka, Japan; and from the Tokyo Biochemical Research Foundation, Tokyo, Japan (to Dr Ueno).
Received May 23, 2000; revision received August 8, 2000; accepted August 9, 2000.
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