Antisense Oligodeoxynucleotide Inhibition of Vascular Angiotensin-Converting Enzyme Expression Attenuates Neointimal Formation
Evidence for Tissue Angiotensin-Converting Enzyme Function
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.
- gene therapy
- renin-angiotensin system
- antisense oligonucleotides
- hemagglutinating virus of Japan
- Received August 6, 1999.
- Accepted October 26, 1999.
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.
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
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 (1×106) 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
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.
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.
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.
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).
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).
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.
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-β, 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.
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.
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