© 2000 American Heart Association, Inc.
Vascular Biology |
Antisense Oligodeoxynucleotide Inhibition of Vascular Angiotensin-Converting Enzyme Expression Attenuates Neointimal Formation
Evidence for Tissue Angiotensin-Converting Enzyme Function
From the Division of Gene Therapy Science (R.M., Y.K.) and the Department of Geriatric Medicine (R.M., N.T., T.O.), Osaka University Medical School, Suita, Japan, and the Department of Medicine (G.H.G., L.Z., V.J.D.), Harvard Medical School, Brigham and Women’s Hospital, Boston, Mass.
Correspondence to Ryuichi Morishita, MD, PhD, Associate Professor, Division of Gene Therapy Science, Osaka University Medical School, 2-2 Yamada-oka, Suita 565, Japan. E-mail morishit{at}geriat.med.osaka-u.ac.jp
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Abstract |
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Abstract—It has been proposed that vascular angiotensin-converting enzyme (ACE) plays an important role in regulating vascular growth. Indeed, ACE inhibitors have been reported to prevent neointimal formation after vascular injury in a rat carotid artery model. However, classic pharmacological experiments cannot exclude the potential contributions of hemodynamics and the circulating renin-angiotensin system (RAS). In this study, we used antisense oligodeoxynucleotide (ODN) to obtain local blockade of vascular ACE expression without effects on systemic hemodynamics and circulating RAS. To increase the effectiveness of antisense action, we modified the hemagglutinating virus of Japan–liposome ODN delivery method by cotransfection with nuclear protein (high mobility group 1 [HMG-1]) and RNase H. In vitro experiments showed the enhanced efficacy of antisense ODN by cotransfection of HMG-1 and RNase H compared with ODN alone. In vivo transfection of antisense ACE ODNs into intact uninjured rat carotid artery resulted in a significant reduction of vascular ACE activity, and cotransfection of HMG-1 and RNase H showed further reduction. We examined the effects of local blockade of vascular ACE expression on neointimal formation after vascular injury. Transfection of antisense ACE ODNs resulted in the attenuation of neointimal formation, whereas sense and scrambled ODNs did not. Blood pressure, heart rate, and serum ACE activity were not affected by antisense treatment. The magnitude of vascular ACE inhibition correlated with the suppression of the neointimal size. Overall, this study demonstrates that local antisense ODN inhibition of vascular ACE expression attenuates neointimal formation independent of hemodynamics and circulating RAS. The results support the existence of a functional tissue angiotensin system in the rat vessel wall.
Key Words: gene therapy • restenosis • renin-angiotensin system • antisense oligonucleotides • hemagglutinating virus of Japan
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Introduction |
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One of the current controversies in cardiovascular research is the existence of a functional tissue angiotensin system1 2 3 4 in the blood vessel. The presence of a vascular angiotensin system has been reported previously.5 6 7 Furthermore, we have described that vascular angiotensin-converting enzyme (ACE) expression is induced in the intimal smooth muscle cells of rat carotid artery and abdominal aorta in response to vascular injury.8 9 This injury model has provided us with the opportunity to examine the functional role of local angiotensin production. Using a dose range of an ACE inhibitor, we demonstrated that the magnitude of neointimal lesion reduction correlated closely with the degree of vascular ACE inhibition.10 We have interpreted these data as supportive evidence for a functional role of tissue angiotensin. However, the blockade of tissue angiotensin required an extremely high dosage of ACE inhibitors, which was invariably accompanied by the suppression of circulating renin angiotensin and the reduction of systemic blood pressure. Therefore, it is still unclear whether the local blockade of vascular angiotensin can indeed prevent neointimal formation without producing a systemic blockade of circulating renin angiotensin and/or without affecting hemodynamics. Furthermore, ACE inhibition also results in bradykinin accumulation and nitric oxide induction, which may contribute to the inhibition of neointimal hyperplasia.
Recent advances in molecular biology have provided us with the opportunity to study the function of a specific local gene product, such as vascular ACE. Using in vivo gene transfer, we have demonstrated that the overexpression of ACE locally in the uninjured rat carotid artery results in the development of vascular hypertrophy that occurs without an effect on blood pressure.11 In the present report, we used a loss of function approach by using antisense oligodeoxynucleotides (ODNs) to ACE mRNA to examine the function of tissue ACE in the injured rat carotid artery in vivo. In the present study, we used a viral protein-liposome–mediated ODN transfer technique that made use of the liposome and the protein coat of the inactivated Sendai virus (hemagglutinating virus of Japan [HVJ]).12 13 HVJ contains 2 envelope proteins, hemagglutinin-neuraminidase and fusion proteins, which mediate cell attachment and membrane fusion sequentially.14 15 In the present study, we reasoned that if tissue ACE contributes to neointimal formation, we would observe a reduction of lesion formation by antisense ODNs in the absence of any effects on systemic blood pressure or the circulating renin-angiotensin system.
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Methods |
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Oligonucleotides
Phosphorothioate ODNs were synthesized and purified by chromatography on NAP 10 columns (Pharmacia). The sequence of each ODN was as follows: antisense ACE 5'-GCCCCCATGGCGCGGT-3', position -8 to +8 of the rat sequence; sense ACE 5'-ACCGCGCCATGGGGGC-3'. Scrambled ODN (5'-CCGTCGGTACCGGCCG-3') was also used as a negative control. Synthetic ODNs were washed by 70% ethanol, dried, and dissolved in sterile Tris-EDTA buffer (10 mmol/L Tris and 1 mmol/L EDTA). The supernatant was purified over a NAP 10 column (Pharmacia) and quantified by spectrophotometry.16 17
Cell Culture
Neonatal vascular smooth muscle cells (VSMCs) were maintained in
Waymouth media with 5% calf serum (CS), which had previously been
inactivated first at 60°C for 1 hour and then at 58°C
for another hour. (This protocol of heat inactivation abolishes serum
ACE activity.18 19 ) Cells (1x106)
were seeded onto 6-well plates and grown to 60% confluence. Previous
data have shown that the addition of the nonhistone nuclear protein
(high mobility group 1 [HMG-1]) enhances plasmid DNA uptake into the
nucleus20 21 and that RNase H enhances the antisense
effect by degrading antisense-mRNA duplexes.22 23
Accordingly, in the present study, phosphorothioate ODN was
incubated with or without HMG-1 (200 µg ODN and 64 µg HMG-1) plus
RNase H (6 U in liposomes) at 20°C for 1 hour. This complex was then
encapsulated in HVJ-liposome. HVJ-liposome (500 µL) containing 3
µmol/L of encapsulated ODN was then added to the wells and incubated
for 5 minutes at 4°C and 30 minutes at 37°C. After transfection,
fresh medium containing 5% CS was added, and the cells were incubated
in a CO2 incubator. On day 3 or 5 after
transfection, cells were homogenized, and ACE activity was
measured. The enzymatic activity, expressed as
hippuryl-L-histidyl-L-leucine–hydrolyzing
activity per milligram of homogenate protein, was
determined by the modified method of Cushman and Cheung.24
The specificity of the ACE activity was confirmed by complete
inhibition by either quinaprilat or neutralizing antibodies to
ACE.18 19
HVJ-Liposome Preparation
Briefly, phosphatidylserine,
phosphatidylcholine, and cholesterol were mixed in a weight
ratio of 1:4.8:2 to create a lipid mixture. Dried lipid was hydrated in
a balanced salt solution containing ODN. Liposomes were prepared by
shaking and sonication. Purified HVJ (Z strain) was
inactivated by UV irradiation (110 erg ·
mm-2 · s-1) for 3
minutes just before use. The liposome suspension (0.5 mL) was mixed
with HVJ (10 000 hemagglutinating units) in a total volume of 4 mL of
balanced salt solution. The mixture was incubated at 4°C for 5
minutes and for 30 minutes with shaking at 37°C. Free HVJ was removed
from the HVJ-liposome by sucrose density gradient
centrifugation. The top layer of sucrose gradient was
collected for use.16 17 18 19
Cotransfection of HMG-1 and Antisense ACE ODNs
Phosphorothioate ODN was incubated with or without HMG-1 (200
µg ODN and 64 µg HMG-1) at 20°C for 1 hour. Then, 500 µL of
HVJ-liposome (3 µmol/L of encapsulated ODN in liposomes) was
added to the wells and incubated for 5 minutes at 4°C and 30 minutes
at 37°C. After transfection, fresh medium containing 5% CS was
added, and the cells were incubated in a CO2
incubator. On day 3 or 5 after transfection, cell ACE activity was
measured.
Cotransfection of RNase H and Antisense ACE ODNs
Before liposome preparation, purified RNase H (GIBCO)
and/or DNA-RNA hybrids were combined with antisense ACE ODN. The
following procedure was described above. DNA-RNA hybrids were made by
the annealing of poly A(RNA)–poly T(DNA) by polymerase chain reaction.
DNA-DNA [poly A(DNA)–poly T(DNA)] hybrids were used as negative
controls. Briefly, equal amounts of poly A and poly T were mixed. Then
this solution was heated to 80°C for 5 minutes and gradually
decreased to room temperature for making a double strand. HVJ-liposome
(500 µL, 3 µmol/L of encapsulated ODN in liposomes) was added
to the wells and incubated for 5 minutes at 4°C and 30 minutes at
37°C. After transfection, fresh medium containing 5% CS was added,
and the cells were incubated in a CO2 incubator.
On day 3 after transfection, cell ACE activity was measured.
In Vivo Transfer Into Intact Rat Carotid Artery
Male Sprague-Dawley rats (400 to 500 g, Charles River
Breeding Laboratories, Atsugi, Kanagawa, Japan) were
anesthetized with ketamine, and the left common carotid
artery was surgically exposed.11 17 18 A cannula was
introduced into the common carotid via the external carotid artery.
HVJ-liposome complex (200 µL, 10 µmol/L ODN with or without
HMG-1 and RNase H [6 U in liposomes]) was infused into the segment
and incubated for 10 minutes at room temperature. After a 10-minute
incubation, the infusion cannula was removed. After the transfection,
blood flow to the common carotid was restored by release of the
ligatures, and the wound was then closed. For the measurement of
vascular ACE activity, rats were euthanized at 7 days after
transfection. After infusion of PBS, carotid arteries were removed and
dissected free of periadventitial tissues and immediately frozen in
liquid nitrogen. On the day of assay, the vessels were thawed, weighed,
and homogenized in 50 mmol/L
KPO4 (pH 7.5). ACE activity was determined as
described above.24 Vascular ACE level was expressed as
enzymatic activity per milligram of protein.
In Vivo Transfection of Antisense ODNs Into Rat Injured
Carotid Artery
In vivo gene transfer was performed under the following
conditions: vascular injury of the common carotid was produced by the
passage and inflation of a balloon catheter (a 2F Fogarty catheter)
through an arteriotomy in the external carotid artery 3
times.16 17 The injured segment was transiently isolated
by temporary ligatures. HVJ-liposome complex (200 µL) containing
antisense ACE ODN, sense ACE ODN (each at 10 µmol/L with or
without 6 U RNase H and HMG-1 contained in liposomes), or scrambled ODN
(10 µmol/L) with HMG-1 and RNase H was incubated within the
lumen for 10 minutes. Two weeks after injury and transfection, each
carotid artery was processed for ACE activity and morphological study.
For histological analyses, a segment of each
artery was perfusion-fixed with 4% paraformaldehyde
and subsequently processed. Medial and luminal areas were measured on a
digitizing tablet (model 2200, South Micro Instruments) after staining
with hematoxylin.16 17 The medial area was readily
demarcated as the vessel area between the internal and external elastic
laminae. At least 3 individual sections from the middle of the
transfected arterial segments were analyzed.
Animals were coded so that the analysis was performed without
the knowledge of which treatment each individual animal received.
Miscellaneous Measurements
Blood pressure was measured by direct measurement with use of a
catheter inserted into the femoral artery of a conscious animal after
recovery from anesthesia (48 hours after
anesthesia). Serum ACE activity was measured from blood
obtained at this time by use of the assay of Cushman and
Cheung,24 as described earlier in this article.
Statistical Analysis
All values are expressed as mean±SEM. ANOVA with a subsequent
Bonferroni test was used to determine significant differences in
multiple comparisons. A value of P<0.05 was considered
significant.
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Results |
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Modification of Antisense ODNs by HVJ-Liposome Method
Using the HVJ-liposome method, we first performed in vitro transfection of antisense ACE ODNs into neonatal VSMCs, which are known to express high levels of ACE.26 A significant reduction in cellular ACE activity was observed at 5 days after transfection with antisense ACE phosphorothioate ODN (3 µmol/L in liposomes, Figure 1a
). Next, we examined the
effect of cotransfection of the nonhistone nuclear protein HMG-1 and
the antisense ACE phosphorothioate ODN into cultured neonatal VSMCs.
HMG-1 has been reported to bind DNA and transport it into the
nuclei,20 27 28 29 thereby increasing the gene expression of
the transfected plasmid.20 In the present study, we
reasoned that cotransfection of ODN with HMG-1 contained in
HVJ-liposome might enhance the nuclear uptake and prolong the action of
ODN. As shown in Figure 1b, an increased inhibitory
effect resulting from the cotransfection of HMG-1 and antisense ODN was
observed, because a measured decrease in ACE activity could be seen at
5 days after transfection with HVJ-liposome. In contrast, HMG-1 alone
did not affect ACE activity. We also studied the enhanced effect of
HMG-1 on a dose range of antisense ODN. Our results demonstrate that
the cotransfection of antisense ODN with HMG-1 in HVJ-liposome was
consistently better than antisense ODN without HMG-1 (data not
shown).
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indicates antisense
ACE ODNs coupled with HMG-1 but without RNase H; •, antisense ACE
ODNs with HMG-1 and RNase H. *P<0.05 and
**P<0.01 vs sense ACE ODNs (with or without RNase H,
respectively).
Within the nucleus, antisense ODN hybridizes with the target mRNA, and
the resultant hybrid is degraded by RNase H as 1 of the major
mechanisms of antisense ODN action.30 31 32 In mammalian
cells, RNase H activity is rather low compared with activity in the
frog oocyte,22 23 33 and one may expect that the
contribution of RNase H to antisense ODN action to be limited. However,
numerous recent reports have shown significant decreases in transcript
levels of the targeted genes with antisense ODN
treatment.16 17 34 Indeed, our results showed a reduction
of ACE mRNA expression (data not shown). Therefore, we examined whether
the cotransfection of purified RNase H with antisense ACE ODN would
result in an enhanced inhibition of ACE expression. We observed that
cotransfection of RNase H and antisense phosphorothioate ODN resulted
in a significant decrease in ACE activity compared with antisense
phosphorothioate ODN alone at 5 days after transfection into VSMCs in
vitro (Figure 1c
). This increased efficacy was due to the
transfection of additional exogenous RNase H into VSMCs, because we
cotransfected synthetic poly A(RNA)–poly T(DNA) hybrids (DNA-RNA
hybrids), which have been used previously to test the RNase H activity
in a cell-free system,22 but not DNA–DNA ligand. There
was an attenuated decrease in ACE activity produced by antisense
ACE-ODN treatment (Figure 1d). Reduction of ACE mRNA was also
enhanced by cotransfection of RNase H and antisense ACE ODN (data not
shown). To exclude the nonspecific effect of RNase H, cotransfection of
RNase H and methylphosphonate antisense ODN was also performed,
because RNase H can cleave RNA-DNA hybrids of unmodified or
phosphorothioate ODN but not those of methylphosphonate
ODN.22 23 As expected, the increase in the in vitro
inhibitory effect of phosphorothioate antisense ODN by
cotransfection of RNase H and HMG-1 was not observed when
methylphosphonate ODNs were used (antisense ACE without RNase H,
55.2±6.0%; antisense ACE with RNase H, 76.1±9.8%;
P>0.05). A 6 µmol/L concentration enwrapped in
liposomes was used. Values are expressed as percentage inhibition of
ACE activity of representative sense ACE
methylphosphonate ODN–treated VSMCs. Finally, we postulated that the
mechanisms of enhanced effects of HMG-1 and RNase H are synergistic.
Accordingly, we examined the inhibitory effect of
cotransfection of RNase H and antisense ACE ODN coupled with HMG-1. As
shown in Figure 1d, cotransfection of antisense phosphorothioate
ODN with HMG-1 and RNase H resulted in a further significant decrease
in ACE activity 5 days after treatment compared with the transfection
of antisense phosphorothioate ODN coupled with HMG-1 without RNase H.
Cotransfection of RNase H and HMG-1 did not result in any cytotoxic
effect as assessed by microscopic examination.
In Vivo Transfection of Antisense ACE ODNs Into Rat Intact
Vessels
By use of this technology, the enhanced effect of antisense ACE
ODNs by cotransfection of HMG-1 and RNase H was also investigated in
vivo in the intact rat carotid artery. One week after transfection,
vascular ACE activity was measured. Antisense phosphorothioate ACE ODNs
significantly decreased ACE activity compared with sense ODNs
(Figure 2). Cotransfection of
HMG-1, RNase H, and antisense ACE ODN decreased ACE activity further
compared with antisense ACE ODN without HMG-1 and RNase H. There was no
significant difference in ACE activity in the intact, sense
ODN–treated vessels compared with the activity in vessels treated with
sense ODN with HMG-1 and RNase H. No morphometric differences were
observed between antisense versus sense ODN–treated uninjured vessels
(data not shown).
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Effect of Antisense ACE ODNs on Neointimal Formation
We also examined the effect of antisense ACE ODN on the
neointimal formation after balloon injury. Administration
of antisense ODN with HMG-1 and RNase H in HVJ-liposome significantly
decreased vascular ACE activity at 2 weeks after transfection
(scrambled ODN, 19.4±2.3 pmol ·
min-1 · mg
protein-1; antisense ODN, 5.6±1.8 pmol ·
min-1 · mg
protein-1; P<0.01). The
inhibitory effect of antisense ODNs with HMG-1 and RNase H
was not accompanied by the inhibition of serum ACE activity (Table 1). In contrast, the other
treatment groups failed to show any reduction in vascular ACE.
Moreover, administration of antisense ACE ODNs (10 µmol/L)
cotransfected with HMG-1 and RNase H (6 U) resulted in the inhibition
of neointimal formation by 50% (Figures 3a and 3b). Similarly,
neointimal areas were also reduced by antisense ACE ODN
treatment accompanied with the reduction of vascular ACE activity,
whereas no significant changes in medial areas were observed in any
group (Table 2). No effects on
blood pressure or heart rate (Table 1) were observed. Residual
vascular ACE activity in the lesion after treatment exhibited good
correlation with the size of the remaining neointimal
lesion, as shown in Figure 3c. Neither sense ODN nor scrambled
ODN with HMG-1 and RNase H had any effect on the neointimal
formation.
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Discussion |
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Angiotensin II (Ang II), a potent vasoconstrictor, has been shown to possess growth-promoting activity. In vitro, Ang II stimulates vascular smooth muscle growth that is mediated by the induction of autocrine growth factors: platelet-derived growth factor, basic fibroblast growth factor, transforming growth factor-ß, and insulin growth factor.4 6 This function of Ang II in vivo has been suggested by studies using Ang II infusion, ACE inhibitors, and vascular injury.10 35 36 However, the contribution of the direct growth effect of Ang II versus its hemodynamic effect to these in vivo actions cannot be dissected with these approaches. Furthermore, recent data suggest that Ang II is produced in local tissues in addition to the circulating Ang II peptide hormone. This concept of a tissue angiotensin system proposes that a significant portion of the growth response in the vessel wall is due to local Ang II. Controversy exists as to the validity of this hypothesis, because inhibition of the local system without systemic effects has not been possible when the current pharmacological approach is used.
Local vascular injury is a good model for examining the effect of increased local tissue ACE. We have reported that 1 to 2 weeks after balloon angioplasty injury of the rat carotid artery or abdominal aorta, ACE expression is induced in the injured vessel, especially in the neointima.9 The level of vascular ACE and, consequently, Ang II correlated with the size of the neointima.10 To examine directly the importance of local ACE in regulating Ang II production and function, we have previously used in vitro and in vivo gene transfer of ACE into cultured VSMCs and intact vessels.18 19 We have demonstrated that transfection of ACE cDNA into VSMCs in vitro results in a significant increase in ACE activity and in Ang II–mediated cellular hypertrophy.18 19 Transfection of ACE cDNA into rat intact carotid artery results in a significant increase in vascular ACE accompanied by a local angiotensin-mediated hypertrophy.11 Because the transfected segment is exposed to the same blood pressure and neurohormones (including circulating renin, ACE, and Ang II levels) as the control segment, these results are strong evidence for a local ACE effect in Ang II production and, consequently, function. Taken together, these data suggest that in addition to the circulating RAS, local angiotensin production is an important modulator of vascular structure.
In the present study, we examined the role of local blockade of the vascular angiotensin system in the prevention of VSMC accumulation after balloon injury by using antisense ODNs to block the local ACE expression without systemic effects. Hence, we addressed the following questions: (1) Does vascular ACE mediate vascular hyperplasia after balloon injury of the rat carotid artery? (2) Is the inhibition of neointimal formation by anti-renin angiotensin agents due to the blockade of local or circulating renin angiotensin or both? (3) Does local angiotensin have a specific role independent of circulating renin angiotensin and/or hemodynamics? These questions cannot be answered by the current pharmacological approach, because the effect of ACE inhibitors and Ang II receptor antagonists on neointimal formation is accompanied by a parallel inhibition of serum ACE activity and fall in blood pressure.10 35 36
To accomplish efficient transfection of antisense ODNs, we used the
efficient HVJ-liposome method and modified it further by cotransfection
of HMG-1 and RNase H. Antisense ODNs are known to be taken up via
receptor-mediated endocytosis, leading to limitation by lower cellular
uptake and degradation by endocytosis-lysosomal
pathways.30 31 32 Using FITC-labeled ODNs, we have reported
that ODNs encapsulated in HVJ-liposomes can enter directly into the
cytoplasm and immediately into the nucleus, resulting in much less
dosage than direct transfer to achieve the same
effect.13 17 37 This approach takes advantage of the
properties of HVJ, a Paramyxovirus that can fuse with cell
membranes at neutral pH.38 The viral proteins of the
HVJ-liposome complex fuse with the cell surface
membrane,39 and the antisense ODN is consequently
introduced directly into the cytoplasm. The character of the
HVJ-liposome method, ie, membrane fusion, resulted in a marked increase
in the efficiency of antisense ODNs compared with passive uptake. The
accumulation of ODNs into nuclei after entering the cytoplasm has been
reported to be due to passive diffusion,41 because the
existence of a nuclear binding site of ODNs is known.42
Although the knowledge of a nuclear binding site of ODNs is relatively
limited, it is logical to think that the number of binding sites may be
a rate-limiting step for the entrance of ODNs into the nuclei, where
RNase H is postulated to function as a major mechanism of the antisense
ODN effect. Therefore, we thought that cotransfection of HMG-1 with
antisense ODN might enhance the effect by increasing the amount of ODN
that is anticipated to function with RNase H in the nuclei, because
HMG-1 has been postulated to bind DNA and carry it into the nuclei by a
different pathway, the ODN binding site.20 21 As expected,
our data showed that HMG-1 will also facilitate antisense ODN
translocation into the VSMC nucleus because cotransfection of HMG-1 and
antisense ACE ODNs in HVJ-liposome resulted in a significantly greater
inhibition of cellular ACE activity compared with antisense ODN alone
(Figure 1b). However, our present results cannot fully
explain whether the enhancement of HMG-1 on antisense action is due to
the enhanced nuclear translocation of antisense ODNs.
Accordingly, we reasoned that cotransfection of RNase H and ODNs can
also increase the effectiveness of antisense ODNs in neonatal VSMCs.
Because the HVJ-liposome method can also deliver protein besides
plasmid DNA and ODN into cells, we added exogenous excess–purified
RNase H to the preparation and demonstrated that their modification
resulted in an enhanced effect of the antisense ODN. Introduction of
additional exogenous RNase H may overcome the rate limitations of low
endogenous RNase H concentrations. The specificity of
exogenous RNase H action was supported by the demonstration that this
effect was inhibited by cotransfection of the synthetic DNA-RNA hybrid
but not the synthetic DNA-DNA hybrid (Figure 1c). Further
evidence for the specificity of the effect of RNase H is also supported
by the observation that cotransfection of RNase H and methylphosphonate
ODNs had no enhanced effects because methylphosphonate ODNs are not
substrates of RNase H. It has been reported that phosphorothioate ODN
is a better target for RNase H than is unmodified ODN.33
Given the widespread usage of phosphorothioate ODNs, this modification
by RNase H may aid in the application of antisense phosphorothioate
ODNs. Taken together, these results show that these modifications of
antisense strategy are effective and that they overcome the 2
limitations to the success of the antisense technology, ie, high
side effects and low efficiency. The present study demonstrates
that using HMG-1 and RNase H combined with antisense phosphorothioate
ODNs can markedly increase the effect and decrease the dose of
antisense ODNs, although the exact mechanisms of the current
modification of antisense delivery using HMG-1 and RNase H has not yet
been clarified.
Using this improved and efficient delivery method, we examined the transfection of antisense ACE ODNs into balloon-injured rat carotid arteries. As previously described, a single transfection of ODN was sustained at least up to 2 weeks after transfection.17 37 Transfection of antisense ACE ODNs resulted in the attenuation of neointimal formation after vascular injury, whereas transfection of sense and scrambled ODNs did not. Importantly, these changes were not accompanied by any changes in hemodynamics (blood pressure and heart rates) or serum ACE. Our data provide evidence that local vascular ACE plays a role in VSMC accumulation in vivo in injured rat carotid arteries that is independent of hemodynamics (no change in blood pressure) and circulating renin angiotensin (no change in serum ACE activity). It is important to point out that although our data support an important functional role of the local angiotensin system, they do not preclude a contribution of the circulating renin-angiotensin system as well. Alternatively, an increase in locally produced bradykinin might affect neointimal hyperplasia, because ACE is also a rate-limiting step in the bradykinin pathway. Further studies are necessary to elucidate the role of bradykinin in this model. Because the present study was performed to examine the existence and function of the tissue angiotensin system in the rat carotid injury model, it was not designed to address the clinical relevance to human restenosis, which is a condition with complex pathophysiological processes beyond neointimal hyperplasia. Indeed, the results of MERCATOR43 and MARCATOR44 trials of ACE inhibition on human restenosis were negative. However, the problems of dosing and timing of therapy in these human studies preclude a definitive conclusion. Alternatively, the failure of those studies may be due to the presence of non-ACE pathways generating Ang II, because chymase, which generates Ang II, has been reported in humans.45 46 Regardless of these issues, our data provide support for the concept that the production of angiotensin locally can result in altered tissue function and structure. Accordingly, one must view the local angiotensin system in the context of tissue function beyond blood pressure regulation. Finally, the present study demonstrated partial inhibition of neointimal formation, whereas the previous findings that made use of antisense ODNs against cell cycle regulatory genes showed almost complete inhibition.16 17 This discrepancy may be due to the multicomplex of the process in the formation of restenosis. The clinical efficacy of antisense strategy against specific growth factors must be discussed in the future.
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Acknowledgments |
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This study was partially supported by grants from the Japan Health Sciences Foundation, the Mochida Memorial Foundation for Medical and Pharmaceutical Research, the Hoan-sya Foundation, the Japan Cardiovascular Research Foundation, and the Japan Heart Foundation Research Grant; a Grant-in-Aid from the Tokyo Biochemical Research Foundation; and a Grant-in-Aid from the Ministry of Education, Science, Sports, and Culture.
Received August 6, 1999; accepted October 26, 1999.
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