© 2000 American Heart Association, Inc.
Vascular Biology |
In Vivo Evidence of the Importance of Cardiac Angiotensin-Converting Enzyme in the Pathogenesis of Cardiac Hypertrophy
From the Department of Geriatric Medicine (J.H., M.A., R.M., I.K., Y.T., N.T., K.Y., A.M., T.O.) and the Division of Gene Therapy Science (R.M., Y.K.), Osaka University Medical School, Osaka, Japan.
Correspondence to Jitsuo Higaki, MD, PhD, Associate Professor, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamada-oka, Suita 565, Japan.
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Abstract |
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Abstract—Cardiac angiotensin-converting enzyme (ACE) may play an important role in regulating cardiac hypertrophy. Angiotensin II (Ang II) stimulates cardiac hypertrophy as well as the production of extracellular matrix. However, it is still unclear whether Ang II exerts a direct effect on cardiac hypertrophy independent of its effect on blood pressure or the circulating renin-angiotensin system. Although ACE inhibitors and/or Ang II receptor antagonists have regressed cardiac hypertrophy, classic pharmacological experiments cannot exclude the contribution of hemodynamics and the circulating renin-angiotensin system. In vivo gene transfer provides the opportunity of assessing the effects of increased cardiac angiotensin in the intact animal without circulating angiotensin or blood pressure. Therefore, we used a "gain of function" approach to obtain local overexpression of cardiac ACE. Transfection of the human ACE vector into rat myocardium resulted in a significant increase in cardiac ACE activity (P<0.01). More interestingly, morphometry at 2 weeks after transfection revealed a significant increase in the thickness and areas of cardiac myocytes in hearts transfected with the ACE vector (P<0.01). In addition, transfection of the ACE vector also resulted in a significant increase in collagen content (P<0.01). This increase in cardiac hypertrophy was abolished by the administration of perindopril. Local transfection of the ACE vector into the heart did not result in systemic effects such as increased blood pressure, heart rate, or serum ACE activity. In summary, we have demonstrated that increased autocrine/paracrine angiotensin can directly cause cardiac hypertrophy independent of systemic factors and hemodynamic effects. This approach has important potentials for defining the role of autocrine/paracrine substances in cardiovascular disease.
Key Words: remodeling • gene transfer • hypertrophy • angiotensin-converting enzyme • renin-angiotensin system
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Introduction |
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The pathogenesis of cardiovascular diseases such as hypertension, atherosclerosis, and myocardial infarction involves a process of cardiac remodeling associated with increased local expression of biologically active substances that are postulated to play pathophysiological roles. In hypertension, the heart undergoes a process of cardiac hypertrophy that is associated with the activation of a local angiotensin system (the renin-angiotensin system [RAS]).1 2 3 The potential role of autocrine/paracrine mediators such as angiotensin in cardiovascular pathobiology independent from systemic factors has been suggested from indirect evidence: cell culture studies, morphological analysis, and/or systemic administration of antagonists and agonists. To elucidate the role of a specific autocrine/paracrine factor, we have developed an efficient in vivo gene transfer technique to examine the consequences of overexpression of the factor in local tissues in the intact rat. Using this approach, we had previously reported that transfection of human angiotensin-converting enzyme (ACE) into local segment of the carotid artery resulted in vascular hypertrophy independent of the circulating RAS and hemodynamics.4 This approach is particularly powerful because the locally transfected vessel segment can be compared with adjacent untransfected segments as well as the contralateral vessel. Furthermore, the transfected segment is exposed to the same blood pressure and circulating factors as the control vessel. However, the role of ACE within the heart still remains unclear, because it is very difficult to transfect efficiently into cardiac myocytes in vivo. To overcome these issues, we reported a high transfection efficiency with the hemmaglutinating virus of Japan (HVJ, or Sendai virus) liposome method into intact rat hearts.5 6 7 For example, luciferase activity was significantly higher in intact noninfarcted hearts transfected with the luciferase vector by the HVJ liposome method than in intact noninfarcted hearts transfected by direct injection of "naked" plasmid DNA.6 Using this method, we further examined the role of autocrine/paracrine angiotensin as a mediator of cardiac hypertrophy in vivo in this study.
Previous data have demonstrated that angiotensin II (Ang II) can stimulate the protein contents of cardiac myocytes and modulate extracellular matrix.8 9 10 11 Ang II is generated via an enzymatic cascade in which tissue ACE plays a key role.12 13 We postulate that increased cardiac ACE expression induces cardiac hypertrophy via increased local generation of Ang II within the heart. Our results provide the first direct evidence that overexpression of an autocrine/paracrine factor (ie, angiotensin) transfected into the heart in vivo mediates the cardiac remodeling process of hypertension independent of systemic factors or hemodynamic stimuli. In this study, we tested our hypothesis by (1) transfecting the human ACE vector locally into intact rat hearts in vivo and (2) studying the biochemical and physiological consequences of overexpression of ACE within the hearts. Our data demonstrate that increased local expression of ACE within the heart promotes autocrine/paracrine Ang II–mediated cardiac hypertrophy in vivo.
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Methods |
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Construction of Plasmids
The pUC-CAGGS expression vector plasmid (kindly provided by Junichi Miyazaki, Tokyo University, Tokyo, Japan)14 was digested with EcoRI. The EcoRI fragment containing the human truncated ACE vector of RB 35-15, including 2 putative active sites, was inserted into the EcoRI site in this vector by filling EcoRI ends with T4 polymerase.4 15 CAGGS contains the entire envelope region open reading frame consisting of 3 translation initiation codons, which represent the N-termini of the large, middle, and major surface (S) polypeptides downstream of the cytomegalovirus enhancer and the chicken ß-actin promoter. The vector used as a control was pUC-CAGGS, which did not contain the ACE cDNA. The luciferase gene expression vector is driven by the Epstein-Barr virus (EBV) promoter or ß-actin promoter.16
Preparation of HVJ Liposomes
The preparation of conventional HVJ liposomes has been described
previously.4 6 17 18 In brief,
phosphatidylserine, phosphatidylcholine, and
cholesterol were mixed in a weight ratio of 1:4.8:2. The
lipid mixture (10 mg) was deposited on the sides of a flask by removal
of tetrahydrofuran in a rotary evaporator. Dried lipid was hydrated in
200 µL of balanced salt solution (137 mmol/L NaCl, 5.4
mmol/L KCl, and 13 mmol/L Tris-HCl, pH 7.6) containing the
DNA–high-mobility group-1 complex (300 µg:96 µg), which had
previously been incubated at 20°C for 1 hour. 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, containing 10
mg of lipids) was mixed with HVJ (20 000 hemagglutinating units) in a
total volume of 4 mL of balanced salt solution. The mixture was
incubated at 4°C for 10 minutes and then for 30 minutes with gentle
shaking at 37°C. Free HVJ was removed from the HVJ liposomes by
sucrose density gradient centrifugation. The top layer
of the sucrose gradient was collected for use. Cationic HVJ liposome
were made by phosphatidylcholine, cholesterol, and
cholesteryl
N-(dimethylaninoethyl)carbamate
mixed in a weight ratio of 8:4:1.16 19
In Vivo Gene Transfer Into the Heart by the Direct Injection
Approach
Male Sprague-Dawley rats (400 to 500 g; Charles River
Breeding Laboratories, Boston, MA) were anesthetized
with an intraperitoneal injection of sodium
pentobarbital (0.1 mL/100 mg body weight). Rats were intubated
and connected to a respirator. HVJ-liposome complex containing human
ACE or the control vector was carefully injected directly
into the heart with a 30-gauge needle through a left lateral
thoracotomy into multiple sites. One injection volume of HVJ-liposome
complex was 10 µL (0.2 µg of plasmid).5 6 7
These experiments were approved by the Osaka University Animal Use
Committee.
Administration of ACE Inhibitor (Perindopril)
Before transfection, rats received Alzet minipumps (Alza
Inc) implanted intraperitoneally. Untreated
animals received vehicle (1:1, vol/vol, of saline/polyethylene glycol
400), whereas the treated groups received the ACE inhibitor
(perindopril, generously donated by Daiichi Pharmaceutical Company,
Tokyo, Japan) at a dose of 10 mg ·
kg-1 · d-1. This
administration regimen had previously been demonstrated to block the
effects of Ang II in vivo.20 21 The drugs were
administered immediately after transfection with human ACE
or the control vector and continued until the hearts were harvested for
morphometry.
Measurement of ACE Activity
For the measurement of cardiac ACE activity, rats were killed 3
days after transfection. After infusion of PBS, the hearts were removed
and immediately frozen in LN2. On the day of
assay, the hearts were thawed, weighed, and homogenized in
50 mmol/L KPO4 (pH 7.5). ACE activity,
expressed as hippuryl-L-histidyl-L-leucine
hydrolyzing activity of the homogenate, was determined by
the modified method of Cushman and Cheung.22 Cardiac ACE
activity was normalized by expressing activity per milligram of tissue.
The specificity of ACE activity was previously confirmed by its
complete inhibition by either quinaprilat (a specific ACE
inhibitor) or neutralizing antibodies to ACE, as previously
described.4 23
Histological Analyses
For morphological analyses, rat hearts were removed 2
weeks after transfection, after perfusion-fixation with 10%
formaldehyde under physiological pressure (110
mm Hg). The thickness and area of cardiac myocytes were measured on a
digitizing tablet (model 2200, South Micro Instruments) after the
tissue was stained with hematoxylin. At least 5 individual sections
from the hearts were analyzed to avoid the effects of needle
injection, as the area of transfection could be detected throughout the
myocardium owing to the multiple injection sites for the
HVJ liposome method.6 7 Animals were coded so that
the analysis was performed without the investigator’s
knowledge of which treatment each animal had received.
Histological analysis was performed by using a
computerized morphometry system (Nexus 6400, Kashiwagi Research Co) by
individuals who were unaware of the treatment each animal had received.
The reproducibility of the results was assessed. Intraobserver
variability was determined from triplicate measurements performed by 1
observer for all sections. The mean±SD differences among measurements
made by the same observer was 2.2±0.4%. Interobserver variability was
determined from measurements of 10 randomly selected sections performed
by a second observer in addition to the first observer. The difference
between measurements made by the 2 observers was 3.3±0.4%. These
observers were blinded to other data concerning the rats, as well as to
the results of the other observer.
Sirius Red Method for Collagen Staining
Sirius red microscopy detects interstitial collagen,
including types I and III.24 Fresh-frozen sections (6
µm) were rinsed with distilled water and incubated with 0.1% Sirius
red F3BA (Polyscience Inc) in saturated picric acid for 90 minutes.
Sections were rinsed twice with 0.01N HCl for 1 minute and then
immersed in distilled water. After dehydration with 70% ethanol for 30
seconds, sections were coverslipped. The stained sections were observed
under polarized light and photographed with the same exposure time for
each section. Analysis of Sirius red staining was performed
with a computer-based quantitative color image analysis
system.25 Photographs were scanned into a 1Kx1K image
buffer of the Optimas 5.2 image analysis system (Optimas Co). A
color threshold mask for immunostaining was defined to
detect the red color by sampling, and the same threshold was applied to
all specimens. The percentage of the total area with positive color for
each section was recorded.
Analysis of Luciferase Activity
Firefly luciferase activity was measured by using a luciferase
assay system (PicaGene, Toyo-Inki). Rats were killed 4 days after
transfection with the luciferase gene either by direct transfection of
"naked" plasmid or by the HVJ-liposome method via direct injection
into the apex, as described below. The tissue samples (200 mg around
the injection site) were rapidly frozen in LN2
and homogenized in a lysis buffer. The tissue lysates were
briefly centrifuged (3000 rpm, 10 minutes), and 20 µL of
supernatant was mixed with 100 µL of luciferase assay reagents.
Measurements of the luminescent reaction were started 5 seconds after
addition of the sample. The counting lasted for 10 seconds, and the
number of counts in 10 seconds was used for calculation of luciferase
activity.6
Statistical Analysis
All values are expressed as mean±SEM. ANOVA with Duncan’s test
was used to determine the significance of differences in multiple
comparisons. P<0.05 was considered to be statistically
significant.
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Results |
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Overexpression of Cardiac ACE
To evaluate the success of gene transfer, we initially measured cardiac ACE activity. The biological activity of the transgene product was documented by a 2-fold increase in ACE activity within ACE-transfected hearts (Figure 1
). Given
that the administration of ACE inhibitors or Ang II
receptor antagonists resulted in the improvement of cardiac
hypertrophy in hypertension, we hypothesized that increased
local production of ACE would modulate cardiac structure.
Therefore, we performed morphological studies 2 weeks after
transfection. Evidence of local cardiac hypertrophy after
ACE gene transfer persisted for at least 2 weeks after transfection, as
reflected by the increasing thickness and area of cardiac myocytes
(Figure 2) and the increasing cardiac
protein content in ACE vector–transfected hearts compared with
controls. Interestingly, administration of the ACE
inhibitor perindopril inhibited the cardiac
hypertrophy induced by the local overexpression of ACE
(Figure 2a). Morphometric analysis documented that the
thickness of ACE transfected–hearts was significantly increased
compared with control vector–transfected and untransfected hearts
(Figure 2b). Consequently, the area of cardiac myocytes in
ACE-transfected hearts was significantly greater than that of control
vector–transfected hearts (Figure 2c). However, there were no
discernible differences in cell numbers counted in serial sections of
the hearts between ACE-transfected and control vector–transfected
hearts (data not shown). Furthermore, we evaluated the effects of ACE
overexpression on collagen synthesis, as Ang II is well known to
stimulate collagen synthesis, and the accumulation of collagen matrix
deposition is a typical feature of hypertensive cardiac
hypertrophy. We performed Sirius red staining for collagen,
as Sirius red staining when viewed under polarized light identifies
collagen, including types I and III.24 Hearts transfected
with control vector showed positive Sirius red staining near the
injection sites only, demonstrating a low content of
interstitial collagen at baseline (Figure 3). In contrast, hearts transfected with
ACE vector exhibited substantial accumulation of
interstitial collagen around the injection site (Figure 3). Of importance, transfection of the human ACE vector into
intact hearts resulted in a significant increase in collagen matrix
compared with control vector, as assessed by quantitative color image
analysis (P<0.01, Figure 3).
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Importantly, these cardiac changes are independent of the circulating
RAS or hemodynamic changes such as blood pressure,
heart rate, and serum ACE activity (Table 1). Moreover, the morphometric changes
typical of cardiac hypertrophy induced by ACE
overexpression were also abolished by administration of the ACE
inhibitor perindopril. These results revealed that
cardiac-specific transfection produced a novel animal model
overexpressing cardiac ACE without interference from the
circulating RAS or hemodynamic changes, such as blood
pressure, heart rate, and serum ACE activity.
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Comparison of Transfection Efficiency
Given the successful production of a novel cardiac
hypertrophy model, we further modified the gene transfer
techniques with the HVJ liposome method. First, comparison of the
expression of different kinds of promoter was evaluated. We constructed
a luciferase expression vector driven by the ß-actin promoter or the
EBV promoter, as the EBV promoter prolongs transgene
expression.16 Consistent with previous findings,
luciferase activity was markedly increased in hearts transfected with
the luciferase vector driven by ß-actin and using the HVJ liposome
method compared with that produced by direct injection
(P<0.01, Figure 4).
Consistent with the previous observation that the EBV promoter
increases the amount of expressed protein,16 our
study showed a significant increase in luciferase activity in hearts
transfected with the luciferase construct driven by EBV promoter
compared with the ß-actin promoter 4 days after transfection.
Moreover, we also modified the HVJ liposome method by changing the
composition of the lipid. In this study, we modified the liposomes from
the anionic to the cationic type, as previous reports had mentioned
that the cationic liposome HVJ liposome method is more efficient for
transfection in vitro as well as in vivo into various tissues compared
with the conventional (anionic) HVJ liposome complex (see Figure 5a).16 19 Thus, we
examined luciferase expression by using the conventional HVJ liposome
complex and the cationic HVJ liposome method for transfection into
hearts. Unexpectedly, luciferase activity was very low in hearts
transfected with the luciferase vector driven by ß-actin promoter and
using cationic HVJ liposomes, whereas high luciferase activity was
observed with the conventional (anionic) composition (Figure 5b).
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Discussion |
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Cardiovascular hypertrophy is thought to be an adaptive response to hypertension. Although it is well accepted that the mechanical effects of increased blood pressure stimulate cardiac and/or vascular hypertrophy, recent indirect evidence suggests that humoral factors may also play an important role. Ang II can stimulate the protein contents of cardiac myocytes and influence extracellular matrix.8 9 10 11 In vivo data from experimental animals have provided additional support for a direct Ang II contribution.26 27 28 29 A key enzyme in the renin-angiotensin biochemical cascade is ACE, which is expressed diffusely in cardiovascular organs such as the heart and blood vessels.1 2 3 12 13 It has been stated that ACE is not rate limiting in Ang II production, since its total quantity in the body is high. However, several investigators have reported that in vivo increases in local cardiac ACE activity in experimental animals result in parallel increases in tissue Ang I conversion to Ang II and changes in local function.8 26 Indeed, it is reported that pressure-overload cardiac hypertrophy induced by aortic banding induces cardiac ACE gene expression and increased cardiac Ang II production.28 Nevertheless, 2 important questions have not yet been resolved in hypertension and cardiovascular research. First, can the RAS directly mediate cardiac hypertrophy, independent of its blood pressure effect? Second, is local tissue ACE important in regulating local cardiovascular function and in contributing to pathophysiology? To answer these questions, we previously employed in vivo gene transfer technology, since these questions have only been addressed indirectly by evidence derived from in vitro cell culture studies, in vivo systemic infusion of Ang II, and/or administration of ACE inhibi-tors.1 2 3 26 27 28 29 In vivo gene transfer technology has the following advantages: (1) the target gene can be transfected into a local segment, thereby avoiding a systemic effect, and (2) the consequences of local overexpression within the physiological/pathophysiological range of the target gene may be studied. Indeed, luciferase activity was detected in the myocardium only by transfection of the luciferase vector by the HVJ liposome method, whereas luciferase activity was not detected in other tissues (brain, lung, liver, kidney, and testis; M.A. et al, unpublished observations, 1996). In contrast, none of these data are completely convincing owing to the contribution of confounding variables such as hemodynamic effects, cell culture conditions, etc. Indeed, our previous study could demonstrate direct evidence of the contribution of vascular ACE in the pathogenesis of vascular hypertrophy.4 However, none of the other reports provided direct evidence of the involvement of cardiac ACE in the pathogenesis of cardiac hypertrophy.
In this study, we have extended our investigation to ACE gene transfer in vivo into rat hearts. We were able to study the long-term cardiac effects of increased tissue ACE activity. As previously reported, HVJ liposome–mediated gene transfer is an efficient in vivo method that has minimal or no toxicity and provides sustained gene expression for transfection into hearts.5 6 7 Moreover, the present study confirmed the high transfection efficiency of the HVJ liposome method by using 2 different kinds of luciferase construct. Although transgenic technology also provides the opportunity to study specific gene function, this technology has several disadvantages: (1) it is time consuming and costly, (2) the effect of the overexpressed transgene is exerted throughout development, and (3) it is difficult to exclude a potential contribution of the systemic effect of transgene expression. Previous studies have documented the roles of hemodynamic stimuli and systemic neurohumoral factors in cardiac hypertrophy in hypertension.3 12 13 28 The present study documents for the first time that the hypertensive cardiac structural response can be mediated by locally generated factors within the heart. Our study supports the notion that ACE is a key rate-limiting enzyme governing Ang II production in cardiac tissues. The increased local expression of ACE is sufficient to induce Ang II–mediated cardiac hypertrophy, independent of changes in the circulating RAS and hemodynamics. This observation is consistent with previous morphometric analyses of hypertensive hearts documenting hypertrophy.30 31 Ang II has been reported to induce the expressions of proliferative factors such as fibroblast growth factor and platelet-derived growth factor, as well as factors influencing the extracellular matrix, such as transforming growth factor-ß.1 11 12 Therefore, myocyte-derived transforming growth factor-ß induced by increased Ang II within the heart may promote the production of extracellular matrix as well as cardiac hypertrophy. One of the features of cardiac hypertrophy in hypertension is the increase in extracellular matrix, such as collagen. Of particular importance, overexpression of cardiac ACE stimulates the accumulation of collagen matrix deposition as a typical feature of hypertensive cardiac hypertrophy. Increased cardiac Ang II may contribute to the pathogenesis of cardiac hypertrophy in hypertension through the accumulation of collagen matrix. Cardiac hypertrophy in rats transfected with the human ACE vector might be due to the autocrine/paracrine effect of locally produced Ang II, since our previous reports demonstrated that locally produced Ang II in cultured vascular smooth muscle cells transfected with the ACE vector increased protein synthesis in an autocrine/paracrine manner.18 Alternatively, a decrease in locally produced bradykinin might affect cardiac hypertrophy, as ACE is also a rate-limiting step in the bradykinin pathway. Further studies are necessary to elucidate the role of bradykinin in this cardiac hypertrophy model.
On the other hand, in vivo gene transfer into the heart opens up the
possibility of local gene therapy for untreatable cardiac diseases such
as myocardial infarction and cardiomyopathy,
although existing methods have many limitations, such as low efficiency
and/or potential toxicity. Therefore, we have reported the usefulness
of the HVJ liposome method for in vivo gene transfer into the intact
heart. In the present study, we further tried to modify this method
for in vivo gene transfer into the heart by using 2 different
approaches: (1) changing promoter construct and (2) modifying the
lipid composition of the liposomes. As previously, we reported the high
transfection efficiency of the HVJ liposome method.5 6 7
For example, luciferase activity was significantly higher in hearts
transfected with luciferase vector by the HVJ liposome method than in
hearts transfected by direct injection of "naked" plasmid DNA (HVJ,
187 000±52 000 light intensity; naked plasmid, 12 000±2100 light
intensity; P<0.01).6 The present
study also confirmed the high transfection efficiency of the HVJ
liposome method compared with naked DNA transfection. Moreover, our
present data also showed significantly high transgene expression
with the use of the EBV promoter, as viral chromosomes such as the EBV
replicon vector can prolong gene expression.16 Further
usage of the EBV promoter would prolong and enhance transgene
expression for long-term experiments. Second, we modified the
composition of the liposomes from anionic to cationic (Figure 5a
). Previous studies had reported an increase in transfection
efficiency with the use of a cationic HVJ-liposome complex in in vitro
as well as in vivo systems in lung epithelial cells.16 19
Unexpectedly, luciferase expression was very low in hearts injected
with cationic HVJ liposomes, whereas high luciferase activity was
observed by using the conventional HVJ liposomes (anionic type). With
the use of FITC-labeled oligodeoxynucleotides, the
low transfection efficiency of cationic HVJ liposomes was also
confirmed in vivo (M.A. et al, unpublished observation, 1996).
The area of transfection was widespread with the HVJ liposome method,
as fluorescence could be clearly detected in the upper part of
only those hearts transfected by the HVJ liposome method,
consistent with previous reports.7 In contrast,
fluorescence was limited to the immediate area around the
injection sites when the cationic HVJ liposome complex was used.
Moreover, significant myocardial damage with accumulation of
neutrophils and necrosis was also observed at the injection site after
cationic HVJ liposome injection but not with conventional HVJ liposomes
(M.A. et al, unpublished observations, 1996). Although
the present study failed to show the usefulness of a modification
of the conventional HVJ-liposome complex, such modification might be
important for developing efficient gene transfer approaches to the
heart to further the understanding and treatment of
cardiovascular diseases.
Overall, the present findings demonstrate a direct role for the cardiac angiotensin system as a mediator of the cardiac hypertrophy process in hypertension, independent of changes in blood pressure or the endocrine RAS. Moreover, this study of the cardiac angiotensin system serves as a paradigm for the elucidation of the role of other autocrine/paracrine mediators of cardiac remodeling in the pathogenesis of diseases such as myocardial infarction and cardiomyopathy. Our data demonstrate that the localized, in vivo gene transfer technique is a useful experimental tool for the study of autocrine/paracrine factors in complex pathophysiological states in vivo. This approach has broad applicability and is complementary to other methods, such as transgenic technology, in elucidating the biological roles of candidate genes in vivo.
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Acknowledgments |
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This work 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, a 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 (to R.M.). The human ACE cDNA was a kind gift of F. Soubrier (INSERM, France). We wish to thank Shiori Takase for her excellent technical assistance.
Received June 29, 1999; accepted September 8, 1999.
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