(Circulation. 2001;103:1893.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Gene Therapy With Extracellular Superoxide Dismutase Protects Conscious Rabbits Against Myocardial Infarction
From the Cardiology Research Laboratory, Division of Cardiology, University of Louisville and Jewish Hospital Heart and Lung Institute, Louisville, Ky (Q.L., R.B., Y.Q., X.-L.T., Y.G.), and the Department of Biomedical Engineering (B.A.F.), University of Virginia, Charlottesville, Va.
Correspondence to Brent A. French, PhD, Department of Biomedical Engineering, University of Virginia, MR4 Bldg, Room 5021, Charlottesville, VA 22903. E-mail bf4g{at}virginia.edu
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Abstract |
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Background—Extracellular superoxide dismutase (Ec-SOD) may protect the heart against myocardial infarction (MI) because of its extended half-life and capacity to bind heparan sulfate proteoglycans on cellular surfaces. Accordingly, we used direct gene transfer to increase systemic levels of Ec-SOD and determined whether this gene therapy could protect against MI.
Methods and Results—The cDNA for human Ec-SOD was incorporated into a replication-deficient adenovirus (Ad5/CMV/Ec-SOD). Injection of this virus produced a high level of Ec-SOD in the liver, which was redistributed to the heart and other organs by injection of heparin. Untreated rabbits (group I) underwent a 30-minute coronary occlusion and 3 days of reperfusion. For comparison, preconditioned rabbits (group II) underwent a sequence of six 4-minute-occlusion/4-minute-reperfusion cycles 24 hours before the 30-minute occlusion. Control-treated rabbits (group III) were injected intravenously with Ad5/CMV/nls-LacZ, and gene-therapy rabbits (group IV) were injected with Ad5/CMV/Ec-SOD 3 days before the 30-minute occlusion. Both groups treated with Ad5 received intravenous heparin 2 hours before the 30-minute occlusion. Infarct size (percent risk area) was similar in groups I (57±6%) and III (58±5%). Ec-SOD gene therapy markedly reduced infarct size to 25±4% (P<0.01, group IV versus group III), a protection comparable to that of the late phase of ischemic preconditioning (29±3%, P<0.01 group II versus group I).
Conclusions—Direct gene transfer of the cDNA encoding membrane-bound Ec-SOD affords powerful cardioprotection, providing proof of principle for the effectiveness of antioxidant gene therapy against MI.
Key Words: myocardial infarction • ischemia • genes • antioxidants • viruses
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Introduction |
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Considerable evidence indicates that reactive oxygen species (ROS), such as superoxide anion (·O2-) and hydrogen peroxide (H2O2), contribute importantly to myocardial ischemia/reperfusion injury.1 When one examines the role of ROS, it is important to distinguish 2 forms of myocardial ischemia/reperfusion injury: reversible postischemic dysfunction (myocardial stunning) and cell death (myocardial infarction [MI]). Because of the fundamental differences between these 2 processes, pathogenetic and pathophysiological information pertaining to one cannot be extrapolated to the other. Indeed, although the role of ROS in myocardial stunning is widely accepted,2 intense controversy persists regarding whether ROS also participate in lethal ischemia/reperfusion injury (MI).1 A number of studies have shown that intravenous administration of antioxidant enzymes (eg, superoxide dismutase [SOD] and catalase) can reduce infarct size; however, other studies have failed to demonstrate a protective effect.1 The reasons underlying this discrepancy are unknown. We hypothesized that the inability of antioxidant enzymes to access the intracellular space after intravenous administration may have contributed to these variable results, and we postulated that the extracellular isoform of SOD (Ec-SOD), which binds to heparan sulfate proteoglycans on cellular surfaces, might provide for more consistent cardioprotection than freely soluble isoforms of SOD.
Another problem with using antioxidant enzymes to protect against MI is that they need to be given parenterally and have short plasma half-lives. These limitations can potentially be overcome by using gene therapy to create an endogenous source of antioxidant protection. Although numerous studies of ischemia/reperfusion injury have used antioxidant enzymes prepared by recombinant techniques,2 3 4 5 6 7 none has used gene therapy to protect intact animals against MI.
The goal of the present study was to compare the protection against MI afforded by a recombinant adenovirus (Ad5) that overexpresses Ec-SOD with that afforded by the late phase of ischemic preconditioning (PC). Although the underlying mechanisms responsible for cardioprotection may be different, the late PC group was included as an internal positive control against which to compare the efficacy of antioxidant gene therapy. We previously reported that Ad5-mediated gene transfer of Ec-SOD protects against myocardial stunning in conscious rabbits.8 In both the previous and current studies, the liver was targeted for gene transfer to exploit the efficiency with which Ad5 transfects hepatocytes after intravenous injection and to preclude the possibility of an inflammatory response against Ad5 in the heart. A conscious rabbit model of MI9 10 was used to overcome limitations inherent in open-chest preparations that could interfere with the study of ischemia/reperfusion injury.11
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Methods |
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Surgical Preparation
The rabbit model of MI has been described previously.9 10 Briefly, pentobarbital-anesthetized New Zealand White male rabbits (weight 2.1±0.2 kg each) were instrumented under sterile conditions with a balloon occluder around a major branch of the left coronary artery, a 10-MHz pulsed Doppler ultrasonic crystal in the center of the region to be rendered ischemic, and bipolar ECG leads on the chest wall. Rabbits were allowed to recover for at least 14 days after surgery. All rabbits were purchased from Myrtles Rabbitry (Thompson Station, Tenn), and the study was performed under protocols approved by the Animal Care and Use Committee at the University of Louisville in accordance with the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, publication No. [NIH] 86-23).
Infarction Protocol
Throughout the protocol, left ventricular (LV)
systolic wall thickening and ECG were monitored continuously on a chart
recorder. Diazepam (4 mg/kg IP) was administered 20 minutes before the
onset of ischemia to prevent stress. The infarction protocol consisted
of a 30-minute coronary artery occlusion followed by 3 days of
reperfusion
(Figure 1
). Successful occlusions were verified by
observation of ST-segment elevations and changes in the QRS complex on
the ECGs and by paradoxical systolic wall thinning on the ultrasonic
crystal recordings.
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Ischemic PC
Ischemic PC was induced in group II with a sequence
of six 4-minute coronary occlusions interspersed with 4 minutes of
reperfusion performed 24 hours before the 30-minute occlusion
(Figure 1
). The proper execution of the occlusion/reperfusion
protocol was evidenced by a 4- to 5-hour period of myocardial stunning
in each
animal.11
Adenoviral Vectors
The constructions of the nuclear-localized LacZ
reporter virus (Ad5/CMV/nls-LacZ) and the recombinant adenovirus that
expresses the human cDNA encoding
Ec-SOD12 were reported
previously.8 Each viral
isolate was plaque purified, verified by restriction analysis, and
evaluated for its potential to overexpress enzymatic activity. Purified
viral clones were propagated in 293 cells, isolated, concentrated, and
titered by plaque assay according to Graham and
Prevec.13
Expression of Ec-SOD
Ad5-mediated expression of Ec-SOD at the mRNA level
was confirmed by infection of COS cells at an MOI of 10 and
harvesting of the cells 2 days later for Northern analysis. Five
rabbits were used to assess Ec-SOD expression in vivo. One was an
untreated rabbit that exemplified group I, whereas the remaining 4
rabbits were treated with 2x108 pfu/kg of
Ad5/CMV/Ec-SOD 3 days before euthanasia to simulate the gene-therapy
protocol (group IV). Total RNA was extracted from samples by standard
procedures (RNAeasy, Qiagen), separated by electrophoresis, and blotted
onto a nylon membrane. A 32P-labeled
riboprobe specific for human Ec-SOD (nucleotides [nt] 1020 through
138912 ) was prepared with
commercial reagents (Maxiscript, Ambion). Northern blots were imaged
with a Storm 840 phosphorimager (Molecular Dynamics) and quantified
with integrated software.
Experimental Design
Three days before MI, rabbits were randomized to 4
groups. Rabbits in the control-treated group (group III) and the
gene-therapy group (group IV) were injected with
2x108 pfu/kg of recombinant adenovirus via
ear vein
(Figure 1). This amount was chosen because the same dose had
previously protected conscious rabbits against myocardial
stunning.8 In both previous
and current studies, heparin was administered 2 hours before the first
occlusion to release Ec-SOD from the liver. Protamine was injected
before coronary occlusion to reverse the effects of heparin. Both
groups III and IV were subjected to this same regimen (ie, Ad5
injection 3 days before coronary occlusion, heparin [2000 U/kg IV] 2
hours before coronary occlusion, and protamine [10 mg/kg IV] over the
last 8 minutes before coronary occlusion).
Measurement of Region at Risk and Infarct
Size
At the conclusion of the study, rabbits were treated
with heparin (2000 U/kg IV), anesthetized with sodium pentobarbital (50
mg/kg IV), and euthanatized with a bolus of KCl. The heart was excised,
mounted on a Langendorff apparatus, and washed extensively with
heparinized saline (50 U/mL) to remove residual Ec-SOD. The repeated
heparin treatments, in combination with the 3-day reperfusion period,
helped to ensure accurate infarct size determinations by
triphenyltetrazolium chloride (TTC)
histochemistry.14 The size
of the ischemic-reperfused region (region at risk) was determined by
ligation of the coronary artery at the site of the previous occlusion
and perfusion of the aortic root for 2 minutes with a 5% solution of
phthalo blue dye at a pressure of 70
mm Hg.9 10 The
heart was then cut into 6 to 7 transverse slices, which were incubated
for 10 minutes at 37°C in a 1% solution of TTC in phosphate buffer
(pH 7.4). All atrial and right ventricular tissues were removed, after
which the slices were weighed, fixed in 10% neutral buffered
formaldehyde, and photographed. The resulting 35-mm slides were
projected at 10x magnification, and the borders of the infarcted,
ischemic/reperfused, and nonischemic regions were traced for digital
planimetry with Adobe Photoshop software. Infarct size was calculated
as a percentage of the region at
risk.9 10
Statistical Analysis
Data are reported as mean±SEM. For intergroup
comparisons, data were analyzed by either 1-way or 2-way
repeated-measures ANOVA (time and group), as appropriate, followed by
unpaired Student’s t tests
with the Bonferroni correction. The relationship between infarct size
and risk region size was compared among groups with ANCOVA, with the
size of the risk region as the
covariate.10 The correlation
between infarct size and risk-region size was assessed by linear
regression with the least squares method. All statistical analyses were
performed with the SAS software
system.15
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Expression of Ec-SOD
As shown in Figure 2A
, mRNA from COS cells infected with Ad5/CMV/Ec-SOD
yielded a discrete band when hybridized with a riboprobe specific for
human Ec-SOD mRNA (lane 1). This positive control band corresponded to
abundant message in livers from rabbits treated with Ad5/CMV/Ec-SOD
(lanes 4 to 7). No signal was detected in the liver (lane 3) or the
heart (lane 8) of a normal, untreated rabbit. As anticipated, no human
Ec-SOD mRNA was detected in the hearts of rabbits treated with
Ad5/CMV/Ec-SOD (lanes 9 to 12), even though abundant message was
detected in livers from these same animals (lanes 4 to 7). As
demonstrated previously,8 the
recombinant Ec-SOD protein is expressed in the liver, secreted from
hepatocytes, and then displaced by heparin for transport through the
bloodstream to the heart and other organs. In
Figure 2B, a quantitative analysis of the Northern blot
demonstrates that intravenous injection of Ad5/CMV/Ec-SOD gave rise to
reproducible levels of human Ec-SOD mRNA expression in the livers of
experimental rabbits.
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Exclusions and Arrhythmias
Of the 51 rabbits instrumented, 13 were assigned to
group I, 12 to group II, 15 to group III, and 11 to group IV. Four
rabbits died of ventricular fibrillation during coronary occlusion (2
in group I and 2 in group II). Two rabbits in group III were excluded
owing to technical problems with the postmortem analysis, and 3 rabbits
(1 in group I and 2 in group III) were excluded because of failure of
the balloon occluder. Thus, a total of 10 rabbits completed the
experimental protocol in group I, 10 in group II, 11 in group III, and
11 in group IV. The incidence of ventricular fibrillation during the
30-minute occlusion and of ventricular tachycardia after reperfusion
did not differ significantly between the control and treated
groups.
Blood Pressure and Heart Rate
Blood pressure and heart rate were monitored in 3
rabbits from group III and 3 rabbits from group IV. Measurements were
taken before coronary occlusion (time 0) and at 6, 24, 48, and 72 hours
after reperfusion. As shown in
Figure 3, heart rate was similar between the 2 groups at
each time point. Blood pressure was also similar between groups at
every time point after reperfusion, although it was slightly lower in
group IV rabbits than in group III rabbits at the time of coronary
occlusion. These results are consistent with previous studies
indicating that Ec-SOD has no effect on blood
pressure.4 6
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Region at Risk and Infarct Size
On the day of the 30-minute occlusion, there were no
significant differences in baseline systolic thickening fraction among
the 4 groups (39.7±2.5%, 35.7±5.1%, 35.4±2.5%, and 35.7±2.6% in
groups I, II, III, and IV, respectively). Similarly, there were no
appreciable differences among groups with respect to LV weight, region
at risk, or region at risk as a percentage of LV weight
(Table).
However, mean infarct size was 50% smaller in the ischemic PC group
(group II) than in the untreated group (group I) (28.6±3.2% versus
56.9±5.9% of the region at risk, respectively;
P<0.01;
Figure 4), indicating a late PC effect against MI. In the
gene-therapy group (group IV), the average infarct size was 57%
smaller than in the control-treated group (group III) (25.1±4.3%
versus 58.3±5.0%, respectively;
P<0.01;
Figure 4), indicating that the expression of Ec-SOD (as
opposed to nls-LacZ) was responsible for the marked cardioprotective
effect. The similarity in infarct size between the ischemic PC group
(28.6±3.2%) and the gene-therapy group (25.1±4.3%) indicates that
the protective effect of gene therapy was comparable to that induced by
the late phase of ischemic PC. The similarity in infarct size between
the untreated group (56.9±5.9%) and the control-treated group
(58.3±5.0%) indicates that neither the administration of an
irrelevant adenoviral vector nor the injections of heparin and/or
protamine had significant effects on infarct size.
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In all groups, the size of the infarction was positively and
linearly related to the size of the region at risk
(Figure 5). As
expected,10 the regression
line was shifted to the right in the ischemic PC group compared with
the control groups (groups I and III)
(P=0.05 by ANCOVA;
Figure 5). In the group pretreated with antioxidant gene
therapy (group IV), the regression line was again significantly shifted
to the right compared with the control groups
(P<0.05 by ANCOVA;
Figure 5) and was virtually indistinguishable from that of
the ischemic PC group, indicating both that for any given size of the
region at risk, the infarct size was reduced by antioxidant gene
therapy and that the magnitude of this effect was similar to that
induced by ischemic PC.
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Discussion |
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The use of in vivo gene transfer to elevate systemic levels of therapeutic proteins has considerable potential, particularly if therapeutic levels can be achieved without adverse side effects. The present study demonstrates that gene therapy with Ec-SOD protects conscious rabbits against MI, indicating that this approach can be effectively used to enhance endogenous antioxidant defenses and limit lethal myocellular injury during ischemia/reperfusion in vivo. Although many studies have found that antioxidant enzymes can protect the heart against ischemia/reperfusion injury (reviewed in Bolli1 ), to the best of our knowledge, this is the first demonstration that the heart can be protected against MI with the use of antioxidant gene therapy, which can potentially provide a sustained supply of antioxidant protein(s). The present study complements earlier work from our laboratory demonstrating that Ec-SOD gene therapy attenuates myocardial stunning in conscious rabbits.8 Together, these studies suggest that gene therapy with Ec-SOD offers considerable potential for protecting the heart against both reversible (myocardial stunning) and irreversible (MI) ischemia/reperfusion injury. Because Ec-SOD is released into the systemic circulation and because this release can be precisely manipulated with heparin and protamine, these studies also suggest a general methodological approach for controlling gene therapy at the posttranslational level and for simultaneously protecting multiple tissues from ROS-mediated injury.
Methodological Considerations
The conscious rabbit model used in the present study is
well characterized with respect to the infarct-sparing effects of
ischemic PC9 10
and avoids a number of factors that could interfere with the assessment
of postischemic myocardial dysfunction (eg, anesthesia, trauma,
temperature fluctuations, abnormal hemodynamics, elevated
catecholamines, and cytokine
release11 ). Most
importantly, the use of a conscious model avoids the exaggerated
oxyradical formation observed in open-chest
animals,11 which could
possibly overwhelm the antioxidant therapy under
investigation.
The choice of antioxidant enzyme was also a critical factor in the present study. Many previous animal studies examining antioxidant enzymes used continuous intravenous infusion of recombinant Cu/Zn-SOD or Mn-SOD protein and thus examined the function of intracellular enzymes in the extracellular space. However, careful examination of the distribution kinetics of Cu/Zn-SOD indicates that the interstitial levels of this enzyme (rather than the plasma levels) are primarily responsible for protection against myocardial ischemia/reperfusion injury.7 This being the case, it was reasonable to consider an isoform of SOD that has natural affinity for the interstitial space. The selection of Ec-SOD for these studies was also influenced by the fact that it is the only isoform of SOD that is secreted from cells and is therefore uniquely suited for hepatic production and systemic distribution.
The possibility of an inflammatory response against the first-generation Ad5 vector prompted us to target gene transfer to an organ other than the heart. Unlike the heart, the liver has a profound regenerative capacity, and remarkably high frequencies of Ad5-mediated transfection (>90%) can be obtained by intravenous injection without compromising hepatic function.16 Not only is the liver an opportune target for Ad5, but this strategy served to alleviate concerns regarding the possibility of inflammation in the heart and its potential impact on ischemic PC and MI.
Microsphere measurements of collateral flow were not undertaken in the present study. However, pilot experiments in our laboratory have demonstrated that rabbits have negligible capability to recruit collateral flow to ischemic regions of myocardium. Minimal collateral flow in ischemic rabbit myocardium has also been reported by others,17 and thus it is unlikely that differences in collateral flow could account for the cardioprotective effects of either late PC or antioxidant gene therapy.
Previous Studies of the Cardioprotective
Effects of Ec-SOD
Numerous studies have examined whether Cu/Zn-SOD and
Mn-SOD can limit tissue damage after coronary
occlusion.1 However,
comparatively few investigations have examined the effect of Ec-SOD on
myocardial ischemia/reperfusion injury, and fewer still have been
conducted in intact animals. Wahlund et
al3 reported that Ec-SOD
reduced creatine kinase release in rats subjected to 10 minutes of
coronary occlusion and 24 hours of reperfusion. Hatori et
al4 found that retroinfusion
of purified, recombinant Ec-SOD protein into the great cardiac vein
decreased the size of MIs in open-chest pigs subjected to coronary
occlusion. Chen et al6 found
that hearts isolated from transgenic mice overexpressing human Ec-SOD
exhibited enhanced postischemic myocontractile function. This
cardioprotective effect is consistent with our present results;
furthermore, the 5-fold elevation in Ec-SOD levels obtained by
transgenesis in the study by Chen et
al6 is comparable to that
obtained in our rabbit model of gene
therapy.8 The present study
differs from previous
investigations3 4 6
in that we examined Ec-SOD produced as a result of gene therapy, and we
used a conscious animal model of MI. Thus, the present study may bear
some clinical relevance, particularly because gene therapy has the
potential to provide a sustained source of antioxidant enzyme over a
period of months or even years.
Finally, previous work from our laboratory described an
Ec-SOD gene-therapy regimen capable of reducing myocardial stunning by
50%.8 That same study
determined that Ad5/CMV/Ec-SOD (2x108
pfu/kg IV) increased total liver SOD activity by 4.4-fold and total
myocardial SOD activity by 2-fold over normal. Subsequent heparin
administration led to a 2-fold increase in total plasma SOD activity
and increased total SOD activity in the heart by a factor of 5.4-fold
over normal. The present investigation does not formally exclude the
possibility that this dose of Ad5 vector might have been
cardioprotective by itself without heparin injection. However, our
previous pilot studies8
suggested that only an intermediate level of cardioprotection would be
obtained at this low dose of virus without the manipulations involving
heparin and protamine.
Present Study
The present report builds on previous
work8 by applying the same
Ec-SOD gene-therapy regimen in a conscious animal model of MI. As shown
in
Figure 2, Northern analysis of rabbits treated 3 days
previously with Ad5/CMV/Ec-SOD revealed consistent levels of human
Ec-SOD mRNA in liver tissue. As summarized in
Figure 4, the same strategy that protected against
myocardial stunning in our previous
study8 also protected the
heart against irreversible ischemia/reperfusion injury, reducing the
size of MI by >50%. This level of cardioprotection was comparable to
that found in rabbits protected by the late phase of ischemic PC.
Interestingly, the cardioprotective mechanism underlying the late phase
of ischemic PC appears to depend on enhanced production of nitric
oxide.10 Cardioprotection by
Ec-SOD may also involve nitric oxide, because elevations in this enzyme
will decrease ambient levels of
·O2-, thus
protecting endogenous nitric oxide from the diffusion-limited reaction
with ·O2- to form
peroxynitrite.
Taken together with our previous study,8 the finding that Ec-SOD gene therapy affords robust protection against MI implicates ROS as important mediators not only of reversible contractile dysfunction (myocardial stunning) but also of irreversible ischemia/reperfusion injury (MI). Thus, the present observations significantly broaden the potential clinical usefulness of Ec-SOD gene therapy and also have important pathophysiological implications regarding the role of ROS in lethal ischemia/reperfusion injury.
The effectiveness of SOD in limiting infarct size has been questioned repeatedly.1 Indeed, although our results are consistent with previous investigations of Ec-SOD,3 4 6 they are seemingly in contrast with several previous studies of Cu/Zn-SOD and Mn-SOD that failed to detect significant cardioprotection against infarction (reviewed in Bolli1 ). Numerous methodological differences in the experimental design of these studies make it impossible to identify the precise reason(s) for the discrepancy. One plausible explanation involves the properties of the antioxidant enzymes examined. Whereas Cu/Zn-SOD and Mn-SOD are freely diffusible in the plasma compartment and interstitial space within the myocardium, Ec-SOD binds to heparan sulfate proteoglycans18 present on the endothelial glycocalyx, in the extracellular matrix, and on the sarcolemma of cardiomyocytes, thereby providing effective protection against ·O2- in the interstitium and on vulnerable cellular surfaces. These strategic locations of the enzyme may enable it to inactivate ·O2- before it can cause extensive tissue damage. The failure of previous studies to detect a protective action of Cu/Zn-SOD and Mn-SOD against MI may have been caused, at least in part, by the relative inability of these proteins to gain access to injurious ·O2-.
Conclusions
The present study demonstrates that gene therapy can be
used to protect conscious rabbits from MI with the use of a single
antioxidant enzyme (Ec-SOD). The efficacy of Ec-SOD in protecting the
myocardium against ischemia/reperfusion injury contrasts with the lack
of consistent protection observed with Cu/Zn-SOD or Mn-SOD and might be
attributed to the extended half-life and/or the extracellular-binding
properties of this unique antioxidant
enzyme.18 The present
results not only implicate ROS in the genesis of lethal
ischemia/reperfusion injury in the conscious animal but also have
significant clinical implications for the development of novel
cardioprotective
strategies.
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Acknowledgments |
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This study was supported in part by a medical research grant (96-413S) from the Jewish Hospital Foundation, Louisville, Ky (Dr French), and by NIH grants R01HL-58582 (Dr French), R01HL-43151 (Dr Bolli), and R01HL-55757 (Dr Bolli).
Received August 21, 2000; revision received October 31, 2000; accepted November 1, 2000.
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J. W. Park, W.-N. Qi, Y. Cai, I. Zelko, J. Q. Liu, L.-E. Chen, J. R. Urbaniak, and R. J. Folz Skeletal muscle reperfusion injury is enhanced in extracellular superoxide dismutase knockout mouse Am J Physiol Heart Circ Physiol, July 1, 2005; 289(1): H181 - H187. [Abstract] [Full Text] [PDF]
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N. Hattan, D. Warltier, W. Gu, C. Kolz, W. M. Chilian, and D. Weihrauch Autologous vascular smooth muscle cell-based myocardial gene therapy to induce coronary collateral growth Am J Physiol Heart Circ Physiol, August 1, 2004; 287(2): H488 - H493. [Abstract] [Full Text] [PDF]
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Y. Xu, S. J. Armstrong, I. A. Arenas, D. J. Pehowich, and S. T. Davidge Cardioprotection by chronic estrogen or superoxide dismutase mimetic treatment in the aged female rat Am J Physiol Heart Circ Physiol, July 1, 2004; 287(1): H165 - H171. [Abstract] [Full Text] [PDF]
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Y. Chu, S. Iida, D. D. Lund, R. M. Weiss, G. F. DiBona, Y. Watanabe, F. M. Faraci, and D. D. Heistad Gene Transfer of Extracellular Superoxide Dismutase Reduces Arterial Pressure in Spontaneously Hypertensive Rats: Role of Heparin-Binding Domain Circ. Res., March 7, 2003; 92(4): 461 - 468. [Abstract] [Full Text] [PDF]
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S. P. Jones, M. R. Hoffmeyer, B. R. Sharp, Y.-S. Ho, and D. J. Lefer Role of intracellular antioxidant enzymes after in vivo myocardial ischemia and reperfusion Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H277 - H282. [Abstract] [Full Text] [PDF]
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M. O. Laukkanen, A. Kivela, T. Rissanen, J. Rutanen, M. K. Karkkainen, O. Leppanen, J. H. Brasen, and S. Yla-Herttuala Adenovirus-Mediated Extracellular Superoxide Dismutase Gene Therapy Reduces Neointima Formation in Balloon-Denuded Rabbit Aorta Circulation, October 8, 2002; 106(15): 1999 - 2003. [Abstract] [Full Text] [PDF]
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S. Hoshida, N. Yamashita, K. Otsu, and M. Hori The importance of manganese superoxide dismutase in delayed preconditioning: Involvement of reactive oxygen species and cytokines Cardiovasc Res, August 15, 2002; 55(3): 495 - 505. [Abstract] [Full Text] [PDF]
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