doi: 10.1161/01.RES.0000065441.72685.29
© 2003 American Heart Association, Inc.
Molecular Medicine |
Gene Therapy With Inducible Nitric Oxide Synthase Protects Against Myocardial Infarction via a Cyclooxygenase-2–Dependent Mechanism
From the Division of Cardiology (Q.L., Y.G., Y.-T.X., S.D.P., W.-J.W., Y.Z., R.B.), University of Louisville, the Jewish Hospital Heart and Lung Institute, Louisville, Ky; The Johns Hopkins University School of Medicine (C.J.L.), Baltimore, Md; and Genetic Therapy, Inc (S.C.S.), Gaithersburg, Md.
Correspondence to Roberto Bolli, MD, Division of Cardiology, University of Louisville, Louisville, KY 40292. E-mail rbolli{at}louisville.edu
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Abstract |
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Although the inducible isoform of NO synthase (iNOS) mediates late preconditioning (PC), it is unknown whether iNOS gene transfer can replicate the cardioprotective effects of late PC, and the role of this protein in myocardial ischemia is controversial. Thus, the cDNA for human iNOS was cloned behind the Rous sarcoma virus (RSV) promoter to create adenovirus (Ad) 5/iNOS lacking E1, E2a, and E3 regions. Intramyocardial injection of Ad5/iNOS in mice increased local iNOS protein expression and activity and markedly reduced infarct size. The infarct-sparing effects of Ad5/iNOS were at least as powerful as those of ischemic PC. The increased iNOS expression was associated with increased cyclooxygenase-2 (COX-2) protein expression and prostanoid levels. Pretreatment with the COX-2–selective inhibitor NS-398 completely abrogated the infarct-sparing actions of Ad5/iNOS, demonstrating that COX-2 is an obligatory downstream effector of iNOS-dependent cardioprotection. We conclude that gene transfer of iNOS (an enzyme commonly thought to be detrimental) affords powerful cardioprotection the magnitude of which is equivalent to that of late PC. This is the first report that upregulation of iNOS, in itself, is sufficient to reduce infarct size. The results provide proof-of-principle for gene therapy against ischemia/reperfusion injury, which increases local myocardial NO synthase levels without the need for continuous intravenous infusion of NO donors and without altering systemic hemodynamics. The data also reveal the existence of a close coupling between iNOS and COX-2, whereby induction of the former enzyme leads to secondary induction of the latter, which in turn mediates the cytoprotective effects of iNOS. We propose that iNOS and COX-2 form a stress-responsive functional module that mitigates ischemia/reperfusion injury.
Key Words: myocardial infarction • adenovirus • mouse • prostaglandins • nitric oxide synthase
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Introduction |
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Unlike the early phase of ischemic preconditioning (PC), which protects against myocardial infarction but not against myocardial stunning, the late phase of PC protects against both infarction and stunning.R1-126656 1,2 Because of this, and because of its sustained duration, late PC may have considerable clinical relevance.R2-126656 2,3 In particular, therapeutic exploitation of this endogenous cardioprotective mechanism may offer a novel approach to the alleviation of myocardial ischemia/reperfusion injury in patients with coronary artery disease.3
One plausible strategy for developing PC mimetic therapies would be to upregulate the protein(s) responsible for the salubrious actions of late PC. Considerable evidence indicates that the cardioprotective effects observed 24 hours after the PC stimulus are mediated by the inducible isoform of NO synthase (iNOS).R2-126656 R4-126656 2,4,5 Specifically, it has been demonstrated that both ischemic PC and pharmacological PC upregulate iNOS, and that pharmacological or genetic ablation of iNOS activity results in abrogation of the beneficial effects of late PC.R4-126656 R5-126656 R6-126656 R7-126656 R8-126656 R9-126656 R10-126656 R11-126656 R12-126656 R13-126656 4–14 iNOS plays an obligatory role not only in the delayed cardioprotection elicited by ischemia, but also in that induced by physical exercise13 and by various pharmacological agents (adenosine A1 receptor agonists,R10-126656 R11-126656 10,11,14 opioid 
1 receptor agonists,11 and endotoxin derivatives9), indicating that this enzyme is a common effector of cardioprotection in response to different stimuli.R2-126656 R4-126656 2,4,5 The central role of iNOS in the shift of the heart to a preconditioned (defensive) phenotype supports the novel idea that augmenting iNOS expression in the myocardium could be a therapeutically useful approach. However, it remains unknown whether upregulation of iNOS via gene therapy is able to replicate the cardioprotective effects of late PC. The traditional perception of iNOS is that of a cytotoxic enzyme.R15-126656 15,16 Furthermore, it is possible that enhancing iNOS activity, in itself, may not be sufficient to effect cardioprotection because late PC is mediated by at least two coinduced proteins (viz, iNOS and cyclooxygenase-2 [COX-2]),R17-126656 R18-126656 R19-126656 17–20 both of which are necessary for the preconditioned phenotype to become manifest.2 It is also possible that an increased tissue content of iNOS protein may not translate into increased iNOS activity because of insufficient availability of essential cofactors such as tetrahydrobiopterin (BH4).R16-126656 R21-126656 16,21,22 In addition, some studies have failed to demonstrate a protective role of iNOS during late PC,23 and others even suggest a detrimental role in acute myocardial infarction.R24-126656 24,25 To date, no investigation has examined whether upregulation of iNOS in itself, in the absence of the other cellular adaptations associated with late PC, is cardioprotective.
The primary objective of the present study was to determine whether increasing the expression and activity of iNOS by in vivo gene transfer confers resistance to lethal ischemic injury. An additional objective was to investigate the role of COX-2 (an obligatory comediator of late PCR17-126656 17,18) in iNOS-dependent protection. We utilized an adenovirus 5 (Ad5) vector with deletions of the E1, E2a, and E3 regions, which has been shown to result in attenuated inflammatory responses compared with first-generation (E1- or E1/E3-deleted) vectors.R27-126656 27,28 A well-established murine model of infarction was used, in which fundamental physiological variables that may impact on infarct size are carefully controlled and measurements of infarct size are highly reproducible.R8-126656 R18-126656 R29-126656 8,18,29,30 Our results demonstrate, for the first time, that increasing regional myocardial NO synthase (NOS) activity via iNOS gene transfer results in significant cardioprotection the magnitude of which is equivalent to that afforded by the late phase of ischemic PC, without perturbation of systemic hemodynamics. Furthermore, we show that the beneficial effects of iNOS gene therapy are associated with, and dependent on, increased COX-2 activity.
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Materials and Methods |
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Adenoviral Vectors
Recombinant adenoviral vectors deleted in the E1, E2a, and E3 regions and carrying either a nuclear targeted ß-galactosidase reporter gene (Ad5/LacZ) or the human iNOS gene (Ad5/iNOS) were constructed as previously described.28
In Vivo Gene Transfer
Anesthetized ICR mice received an intramyocardial injection in the anterior left ventricular (LV) wall of adenoviral vehicle (PBS2+/1% sucrose [vehicle group]), Ad5/LacZ (1x107 pfu [Ad5/LacZ group]), or Ad5/iNOS (1x107 pfu [Ad5/iNOS group]). Three days later, mice underwent the infarction protocol described below (online Figure 1).
Coronary Occlusion/Reperfusion Protocol
The murine model of myocardial ischemia and reperfusion has been described in detail.R8-126656 R18-126656 8,18,29 In all groups, myocardial infarction was produced by a 30-minute coronary occlusion followed by 4 hours of reperfusion29 (online Figure 1). Group I (sham-preconditioned group) underwent a thoracotomy with 1 hour of open-chest state without coronary occlusion/reperfusion. Group II (late PC group) was preconditioned with a sequence of six 4-minute occlusion/4-minute reperfusion cycles 24 hours before the 30-minute occlusion (online Figure 1). Groups III, IV, and V received an intramyocardial injection of vehicle (vehicle group), reporter virus (Ad5/LacZ group), or Ad5/iNOS (Ad5/iNOS group), respectively, as described above, 3 days before the 30-minute occlusion. Groups VI and VII received an intramyocardial injection of Ad5/LacZ or Ad5/iNOS, respectively, 3 days before the 30-minute coronary occlusion; 30 minutes before the coronary occlusion, they were given the selective COX-2 inhibitor NS-398 (5 mg/kg IP [Ad5/LacZ+NS-398 and Ad5/iNOS+NS-398, respectively]).
Postmortem Tissue Analysis
At the conclusion of the study, the occluded/reperfused vascular bed and the infarct were identified by postmortem perfusion of the heart with phthalo blue dye and triphenyltetrazolium chloride (TTC)R8-126656 R18-126656 8,18,29 (Figure 1).
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Western Immunoblotting Analysis
The expression of iNOS, endothelial NOS (eNOS), neuronal NOS (nNOS), COX-1, and COX-2 was assessed by standard SDS/PAGE Western immunoblotting techniques.R8-126656 8,17 In all samples, the content of NOS or COX protein was expressed as a percentage of the corresponding protein in the Ad5/LacZ group (viral control group).
Measurement of iNOS Activity, Nitrite and Nitrate (NOx), and Prostaglandins (PGs)
Calcium-independent NOS activity (iNOS activity) was determined by measuring the conversion of [14C]L-arginine to [14C]L-citrulline as previously described.8 Myocardial and serum levels of NOx were measured as previously described.8 The myocardial content of PGD2, PGE2, PGF2
, and 6-keto-PGF1 (the stable metabolite of PGI2) was determined using enzyme immunoassay kits.17
Histology and Immunohistochemistry
These methods are described in the online supplement available at http://www.circresaha.org.
Statistical Analysis
Data are reported as mean±SEM. The statistical analysis is detailed in the online supplement.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
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Results |
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A total of 237 mice were used in this study (84 for the infarct size studies and 153 for biochemical analyses). The number of mice excluded in the infarct size studies and the reasons for exclusion are detailed in online Table 1.
iNOS Gene Expression In Vitro
The ability of Ad5/iNOS to induce expression of functionally competent protein was verified in transfected COS-7 cells (online Figure 2).
iNOS Gene Expression In Vivo
In the LacZ group, the region of gene expression (as assessed by X-gal staining) was 17.5±3.3% of the region at risk (n=6) (Figures 1 and 2A). Three days after intramyocardial injection of vehicle (PBS2+/1% sucrose [vehicle group]), Ad5/LacZ (Ad5/LacZ group), or Ad5/iNOS (Ad5/iNOS group), the myocardium surrounding the site of injection (
10 mg) was harvested for measurement of iNOS protein expression, iNOS activity, and NOx levels. Western immunoblotting detected expression of iNOS protein in both the vehicle and the Ad5/LacZ groups (Figure 3, top panel), consistent with previous studies showing that iNOS is constitutively present in murine myocardium.8 However, the expression of iNOS was significantly increased (2.5-fold in the cytosolic fraction and 2.4-fold in the membranous fraction) in mice transfected with Ad5/iNOS as compared with mice transfected with reporter virus (Ad5/LacZ) (Figure 3, top panel). iNOS activity was also significantly increased in mice transfected with Ad5/iNOS (4.2-fold greater than in the Ad5/LacZ group; Figure 3, middle panel), indicating that the transgenic protein was functionally competent. In contrast, eNOS protein expression did not change (online Figure 3). No nNOS immunoreactivity was detected (data not shown). Consistent with the measurements of iNOS activity, the myocardial levels of NOx (the stable oxidation products of NO) were significantly increased (+56%) 3 days after injection of Ad5/iNOS compared with the Ad5/LacZ group (Figure 3, bottom panel). However, NOx levels in the serum were indistinguishable between the two groups (Figure 3, bottom panel), indicating that the source of NO was in the myocardium. This was confirmed by Western measurements of iNOS protein expression, which was found to be unchanged in lung, liver, kidney, and spleen (online Figure 4). The fact that iNOS expression or activity did not increase in the Ad5/LacZ group (Figure 3) demonstrates that the upregulation of iNOS observed in the Ad5/iNOS group cannot be ascribed to nonspecific factors associated with gene transfer (eg, trauma, local inflammatory reaction, etc).
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As illustrated in Figures 2B and 2D, robust iNOS immunoreactivity was present in myocytes of hearts transfected with Ad5/iNOS. No iNOS immunoreactivity was noted in sections incubated with nonimmune serum (not shown). Histological analysis with hematoxylin and eosin (H&E) staining revealed no evidence of inflammation around the myocytes transduced with Ad5/iNOS (Figure 2F), except for minor cellular infiltration along the needle track (data not shown).
Effects of iNOS Gene Transfer on COX-2 Protein Expression and PG Content
Three days after iNOS gene transfer, the gene-transduced region exhibited a significant increase in COX-2 protein expression (3.4-fold versus the Ad5/LacZ group [Figure 4]) and in the tissue levels of PGE2 and 6-keto-PGF1 (the stable metabolite of PGI2) (+92% and +55%, respectively, versus the Ad5/LacZ group [Figure 4]). In contrast, myocardial PGF2 and PGD2 levels did not change significantly (Figure 4). COX-1 protein expression was unchanged (online Figure 5). As illustrated in Figures 2C and 2E, intense COX-2 immunoreactivity was found in cardiac myocytes within the gene-transduced region and the spatial distribution of COX-2 expression (Figure 2C) coincided with that of iNOS expression (Figure 2B), indicating that COX-2 and iNOS were co-upregulated in the same cells. COX-2 immunoreactivity was also observed in endothelial cells, but this signal was similar in transduced and nontransduced myocardium (data not shown).
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Baseline Determinants of Infarct Size
By experimental design,29 rectal temperature remained within a narrow, physiological range (36.7°C to 37.3°C) in all groups. The ventilatory settings have previously been shown to result in normal arterial blood gases and pH.29 Three days after gene transfer, mean arterial pressure (measured by carotid artery cannulation in a subset of mice) was similar in the Ad5/iNOS and Ad5/LacZ groups (91.3±3.6 mm Hg [n=6] versus 92.8±2.1 mm Hg [n=6], respectively). Similarly, heart rate did not differ among the seven groups on the day of the 30-minute coronary occlusion (online Table 2). Thus, fundamental physiological parameters that may impact on infarct size were similar in all groups of mice.
Infarct Size
There were no significant differences among the seven groups with respect to LV weight or weight of the region at risk (online Table 3). To better evaluate the efficacy of iNOS gene therapy, the infarct-sparing effects of Ad5/iNOS injection were compared with those of the late phase of ischemic PC. When mice were preconditioned with six cycles of 4-minute coronary occlusion/4-minute reperfusion cycles 24 hours before the 30-minute coronary occlusion, infarct size was reduced by an average of 51% versus sham-preconditioned mice, indicating a late PC effect (Figure 5). In mice given Ad5/LacZ, infarct size did not differ from that measured in mice given vehicle or sham PC (Figures 1 and 5). Thus, neither the administration of an irrelevant adenoviral vector nor the intramyocardial injection itself modified the extent of myocardial cell death. However, in mice that received Ad5/iNOS, the mean infarct size was 67% smaller than that in the control virus group (Ad5/LacZ group) (Figures 1 and 5), demonstrating that the increased expression of iNOS was associated with profound cardioprotection. The average infarct size observed in the Ad5/iNOS group was even smaller than that observed in the late PC group (Figure 5), indicating that the infarct-sparing effects of iNOS gene therapy were at least equivalent to those of the late phase of ischemic PC. Pretreatment with the selective COX-2 inhibitor NS-398 did not modify infarct size in Ad5/LacZ-treated mice (Figure 5) but completely abrogated the infarct-sparing actions of Ad5/iNOS injection (Figure 5). As expected,R18-126656 18,29 in all groups the size of the infarction was positively and linearly related to the size of the region at risk (online Figure 6); analysis of these data by ANCOVA confirmed the results obtained by comparing average infarct sizes (see legend to online Figure 6).
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Discussion |
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Although iNOS is a necessary mediator of late PC,R2-126656 R4-126656 2,4,5 several considerations seem to argue against the idea that isolated augmentation of iNOS activity is sufficient to mimic this cardioprotective adaptation. First, late PC is a polygenic adaptation to stress that involves the coordinated activation of multiple signaling and effector proteins2; consequently, upregulation of a single enzyme (such as iNOS) may not suffice to recapitulate this phenotype. Second, iNOS requires a number of cofactors, including BH4,16 and it is unclear whether the endogenous BH4 biosynthetic capacity of the myocardium is sufficient to sustain enhanced iNOS activity. In certain cell types, iNOS gene transfer does not result in increased iNOS activity unless exogenous BH4 is supplemented.R21-126656 21,22 Finally, some investigators have not found evidence for a cardioprotective function of iNOS (ie, pharmacological inhibitors of iNOS have been reported to reduce infarct size24 and genetic ablation of iNOS has been reported to have no effect on adenosine-induced late PC23). To date, no study has investigated whether iNOS gene therapy is cardioprotective.
Salient Findings
To address this question, we examined the effects of iNOS gene transfer by using an Ad5 vector with deletions of the E1, E2a, and E3 regions. Immunoblotting and biochemical analyses demonstrated that intramyocardial injection of Ad5/iNOS resulted in a significant local increase in iNOS protein expression, iNOS activity, and NOx levels. This upregulation of iNOS activity was associated with a profound reduction in infarct size, providing the first direct evidence for a cardioprotective function of iNOS. Furthermore, the increased iNOS expression was accompanied by increased expression and activity of COX-2, and administration of a COX-2–selective inhibitor abrogated the cardioprotective effects of iNOS gene therapy, demonstrating that COX-2 is an obligatory downstream effector of iNOS-dependent cardioprotection.
Previous studies have indicated a cytoprotective role of iNOS in myocardium preconditioned by ischemia, exercise, or pharmacological agents.R2-126656 R4-126656 R5-126656 R6-126656 R7-126656 R8-126656 R9-126656 R10-126656 R11-126656 R12-126656 R13-126656 2,4–14 However, to our knowledge, this is the first demonstration that upregulation of iNOS expression in itself (in the absence of the other cellular adaptations induced by ischemic or nonischemic PC stimuli) is sufficient to reduce the extent of myocardial infarction. This is also the first report that iNOS activity induces COX-2 protein expression, which is essential for the infarct-sparing actions of iNOS. The latter finding supports the novel idea that iNOS and COX-2 form a tightly coupled functional module designed to mitigate ischemia/reperfusion injury. Taken together, the present results have conceptual implications for our understanding of the function of iNOS and COX-2 in the heart as well as practical implications for the development of therapeutic strategies designed to limit tissue destruction during myocardial ischemia.
Methodological Considerations
The Ad5 vector used herein, with deletions in the E1, E2a, and E3 regions, effects gene transfer and expression with efficiency comparable to that of the first-generation (E1- or E1/E3-deleted) vectors,28 but offers the advantage that the defective DNA binding protein encoded by E2a is incapable of contributing to the activation of the major late gene promoter.27 Because adenoviral late gene products are not expressed, the host immune response toward the transduced cells is attenuated, resulting in an improved toxicity profile and prolonged transgene expression after in vivo delivery.28 In terms of clinical safety, these vectors should have reduced potential for cell transformation or for recombination resulting in replication-competent adenovirus.R26-126656 26,31 Accordingly, E1-, E2a-, and E3-deleted vectors may mediate efficient long-term in vivo gene transfer with greater safety than previously utilized vectors.28
iNOS Gene Therapy for Cardioprotection
The choice of the gene to be tested in this study (iNOS) was predicated on several considerations. First, iNOS is an obligatory mediator of late PC.R2-126656 R4-126656 2,4,5 Second, extensive evidence supports a cytoprotective role of NO during myocardial ischemia/reperfusion.5 Third, NO is highly diffusible. Transfer of genes encoding other cardioprotective proteins (eg, heat shock proteins, antioxidant enzymes, or antiapoptotic proteins) could in principle be beneficial, but the majority of these therapies will be limited by the number of cells that can be targeted. Because the products of these genes are not secreted, only cells expressing the transgene will be affected, which may be prohibitively inefficient, particularly in view of the fact that methods for uniform gene transfer to the heart are still lacking. In contrast, the highly diffusible nature of NO enables this gas to access many cells that do not express the iNOS transgene, thereby amplifying the beneficial effects of iNOS gene transfer. The prostanoids derived from iNOS-dependent COX-2 activity are also diffusible and can amplify the effects of iNOS gene therapy. These concepts are supported by our finding that iNOS gene transfer reduced infarct size by an average of 67% despite the fact that only 18% of the risk region exhibited transgene expression (as assessed by X-gal staining) (Figures 1 and 2A), demonstrating that NO produced in the transduced cells protected a larger adjacent myocardial region. These properties of iNOS gene transfer overcome, at least in part, the major obstacle to gene therapy, ie, the limited number of cardiac cells that can be transduced with currently available methods.
Gene transfer of iNOS could offer significant advantages relative to other NOS isoforms or to NO donors. Compared with eNOS or nNOS, iNOS synthesizes much larger quantities of NO in a sustained manner and functions independently of intracellular calcium fluxes or other stimuli, thereby maintaining tonically augmented myocardial NO levels.16 Therefore, even if iNOS is expressed in relatively few cells, a wide field of cells could be exposed to NO (or to its effectors such as PGs) without the need for the external stimuli required for eNOS or nNOS stimulation. Ischemia/reperfusion injury can be ameliorated by infusion of NO donors.5 However, systemic NO donor therapy is inherently limited by its hemodynamic effects, most notably hypotension, and local delivery is impractical. Oral or transdermal NO donor administration alleviates these problems, but the local myocardial NO levels achieved via these routes may not be sufficient to elicit cardioprotection, and the effects are transient. In contrast, iNOS gene therapy makes it possible to generate sustained high local levels of NO in target tissues without incurring systemic side effects.
A potential problem with NOS gene transfer lies in the ability of targeted cells to produce the cofactor BH4, which is essential for the function of all NOS enzymes.16 However, our finding that transfection with Ad5/iNOS resulted in increased myocardial synthesis of NO (Figure 3, bottom panel) implies that exogenous supplementation of BH4 (or other cofactors) is not necessary for the activity of the transgenic protein.
iNOS as a Cardioprotective Protein
Until recently, the role of iNOS in the cardiovascular system was generally thought to be detrimental because of its involvement in such conditions as septic shock, inflammation, allograft rejection, and cerebral infarction.R15-126656 R16-126656 15,16,32 This concept was challenged by the discovery that iNOS is an obligatory mediator of late PC.4 By demonstrating that iNOS in itself, irrespective of late PC, exerts profound cardioprotective actions, the present study further supports the need to reassess traditional views that attribute a deleterious function to this enzyme in myocardial ischemia. Our results are consonant with the finding that transfection with Ad5/iNOS protects endothelial cells against apoptosis33 and exerts vascular protective actions,R34-126656 34,35 and with the emerging evidence that iNOS is a hypoxia-responsive gene that plays a protective role not only in the heart but also in other tissues (eg, kidney and intestine).5
Our observations are apparently at odds with previous studies,R24-126656 R25-126656 R36-126656 24,25,36,37 which have concluded that iNOS exerts detrimental effects in the setting of myocardial infarction. In two of these studies, iNOS-/- mice exhibited no change in infarct size but were reported to have lower mortality,R36-126656 36,37 improved LV function,36 and higher peak LV developed pressure at maximal (but not intermediate) preload37 compared with wild-type mice. Two investigations have reported a smaller infarct size after administration of the iNOS inhibitor aminoguanidine (AMG) or S-methylisothiourea (SMT).R24-126656 24,25 Interpretation of these results is complicated by the fact that both AMG and SMT have modest selectivity for iNOS versus eNOS/nNOS38 and exert a large number of nonspecific effects.38 On the other hand, numerous studies have found no difference in infarct size or short-term mortality between iNOS-/- and wild-type mice.R8-126656 R9-126656 R10-126656 R11-126656 8–11,13 Major issues that need clarification include the long-term effects of iNOS overexpression, the effects of iNOS in the nonischemic region, and the relative roles of iNOS in myocytes versus inflammatory cells.
Role of COX-2 in iNOS- Dependent Cardioprotection
In the context of ischemia-induced late PC, we have previously shown that the activity of COX-2 is driven by iNOS activity.19 However, it remains unknown (1) whether iNOS modulates cardiac COX-2 activity in the absence of ischemic PC; (2) whether iNOS enhances only the activity of COX-2 or also the abundance of COX-2 protein expression; and (3) perhaps most importantly, whether the increased COX-2 activity is necessary for the cardioprotective effects of iNOS to become manifest. In noncardiac cells, there are conflicting reports regarding the ability of NO to induce COX-2 protein.R39-126656 R40-126656 R41-126656 39–42
In this study we found that the upregulation of iNOS was associated with increased COX-2 protein expression (Figure 4) and increased PGE2 and PGI2 levels (Figure 4), and that the cardioprotection afforded by iNOS gene therapy was ablated by the administration of the COX-2–selective inhibitor NS-398 (Figure 5). These data demonstrate that (1) increased iNOS activity in itself, in the absence of ischemia or any PC stimulus, is sufficient to induce myocardial COX-2 expression in vivo, revealing a new facet of the regulation of COX-2, and (2) increased biosynthesis of COX-2–derived cytoprotective prostanoids, such as PGE2 and PGI2, represents an important mechanism for iNOS-dependent cardioprotection. On the basis of these observations, we propose that there exists a close functional coupling between cardiac iNOS and cardiac COX-2, such that induction of the former enzyme leads to secondary induction of the latter, which in turn mediates the cytoprotective effects of iNOS.
Conclusions
The function of iNOS in the cardiovascular system remains largely enigmatic. Using a physiologically relevant murine model of myocardial infarction, the present investigation provides new insights into the role of this poorly understood protein. In contrast to recent claims that iNOS exacerbates myocardial ischemic damage,R24-126656 R25-126656 R36-126656 24,25,36,37 the data presented herein demonstrate that iNOS gene therapy affords powerful protection against lethal ischemia/reperfusion injury, at least equivalent to that afforded by ischemic PC, and that this effect is mediated via enhanced COX-2 activity.
From a conceptual standpoint, the discovery of a cytoprotective iNOS–COX-2 module expands our understanding of the significance of these two stress-responsive proteins in the heart and challenges existing paradigms. Further studies are warranted to elucidate the molecular interactions between iNOS and COX-2. From a practical standpoint, the present results provide proof-of-principle for the utility of a form of gene therapy against ischemia/reperfusion injury that increases local myocardial iNOS levels without the need for continuous intravenous infusion of NO donors and without altering systemic hemodynamics. This has potential clinical applications, because the transgene encodes human iNOS and the E1-, E2a-, and E3-deleted Ad5 vector has a higher safety profile than first-generation Ad5 vectors. Because of the diffusible nature of NO and prostanoids, efficiency of gene transfer is not as critical with Ad5/iNOS as it would be with other cardioprotective proteins that do not act via secreted/diffusible products. Indeed, a gene such as iNOS that is effective when expressed in low abundance could have distinct advantages. Thus, the present findings provide a rationale for exploring the utility of iNOS gene therapy in protecting against myocardial ischemia/reperfusion injury.
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Acknowledgments |
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This study was supported in part by NIH Grants HL43151, HL55757, HL68088, HL70897, and HL65660.
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Footnotes |
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This manuscript was sent to Eugene Braunwald, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Dr Charles J. Lowenstein received a research grant from Novartis from 1996 to 1999.
Received June 3, 2002; revision received January 9, 2003; accepted February 26, 2003.
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