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B and AP-1 activation and enhances myocardial damage
Children’s Hospital Medical Center, Division of Critical Care, Cincinnati, Ohio 45229, USA
1Correspondence: Division of Critical Care Medicine, Children’s Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229, USA. E-mail: bzingarelli{at}chmcc.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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(TNF-), interleukin 6 (IL-6), and IL-10 were also increased. These events were preceded by degradation of inhibitor 
B (IB), activation of IB kinase complex (IKK) and c-Jun-NH2-terminal kinase (JNK), and subsequently activation of nuclear factor-B (NF-B) and activator protein 1 (AP-1) as early as 15 min after reperfusion. In contrast, iNOS-/- mice experienced 35% mortality after reperfusion. The extensive myocardial injury was associated with marked apoptosis and infiltration of neutrophils whereas expression of HSP70 was less pronounced. Nitrotyrosine formation and plasma levels of nitrite and nitrate were undetectable. TNF- and IL-6 were increased and IL-10 was reduced in earlier stages of reperfusion. Activation of IKK and JNK and binding activity of NF-B and AP-1 were significantly reduced. Thus, we conclude that iNOS plays a beneficial role in modulating the early defensive inflammatory response against reperfusion injury through regulation of signal transduction.—Zingarelli, B., Hake, P. W., Yang, Z., O’Connor, M., Denenberg, A., Wong, H. R. Absence of inducible nitric oxide synthase modulates early reperfusion-induced NF-B and AP-1 activation and enhances myocardial damage.
Key Words: apoptosis • nuclear factor B • activator protein 1 • heat shock protein 70 • nitrotyrosine
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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2
3)
. Current evidence suggests that oxidative stress during reperfusion is an important signal transduction that induces a coordinate trans-activation of genes of proinflammatory and anti-inflammatory mediators including cytokines, adhesion molecules, heat shock proteins, and other cytotoxic or cytoprotective molecules. Such a signal may be mediated at the transcriptional level by a rapid activation of the enhancer elements nuclear factor B (NF-B) and activator protein 1 (AP-1) through interactions with IB kinase (IKK) and c-Jun-NH2-terminal kinase (JNK), respectively (4
5
6
7)
.
Nitric oxide (NO) is an important mediator of biological processes such as vascular homeostasis, neurotransmission, immunity, and inflammation. Under physiological conditions in the cardiovascular system, NO from the constitutive endothelial NO synthase (ecNOS) plays an important homeostatic role. The constitutive release of NO is critical to the preservation of vasodilatation, platelet adhesion and aggregation, microvascular permeability, and smooth muscle cell proliferation. NO also regulates neutrophil recruitment by inhibiting the expression of adhesion molecules in the vascular endothelium and has negative chronotropic and inotropic effects (8)
.
However, an overwhelming production of NO by an inducible NOS (iNOS) has been demonstrated in many inflammatory processes. The precise role of NO generated by iNOS during myocardial ischemia and reperfusion is not completely understood and is under controversial debate. Several studies of in vivo and in vitro experimental conditions of myocardial ischemia and reperfusion injury have demonstrated that pharmacological inhibition of NO synthesis or genetic abolition of ecNOS exerts deleterious effects, whereas treatment with NO donors or superinduction of iNOS attenuates reperfusion-induced arrhythmias and myocardial infarction and improves contractile function (9
10
11)
. In line with these findings, it has been demonstrated that pharmacological or genetic inhibition of iNOS abolishes the cardioprotection afforded by ischemic preconditioning or monophosphoryl lipid A (12
13
14
15)
. In contrast to these findings, however, it has been reported that increased release of NO may contribute to ischemia and reperfusion injury and that NO inhibition may exert cardioprotective effects (16
17
18)
.
Using knockout mice with a targeted disruption of the iNOS gene and control mice with a functional iNOS gene, we investigated the role of endogenous iNOS-derived NO in the development of tissue damage in ischemic hearts after early reperfusion. We determined the extent of myocardial cell apoptosis, neutrophil accumulation, and the release of proinflammatory cytokines such as tumor necrosis factor (TNF-) and interleukin 6 (IL-6) or anti-inflammatory mediators, such as IL-10 and the cardioprotective heat shock protein 70 (HSP70). To gain a better understanding as to the regulatory role of NO, we investigated whether the genetic absence of iNOS may affect the signal transduction mechanisms that trigger reperfusion injury.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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. The chest was opened by a cut along the left side of the sternum through the ribs. The animal was rotated to expose the left ventricle. Ligation proceeded with a 7–0 silk suture passed with a tapered needle underneath the left anterior descending branch (LAD) of the left coronary artery. A 1 mm section of PE-10 tubing was placed atop the vessel and a knot was tied on top of the tubing to occlude the artery. In the heart, the cardiac venous network was clearly visible with a dissection microscope, and no veins were occluded with this maneuver. After 60 min occlusion, reperfusion occurred by cutting the knot on top of the tubing with a surgical blade. Different groups of mice were killed at the end of the ischemia (60 min) or at various times after reperfusion (15, 30, 45, 60, 120, and 240 min). Blood samples were collected. Hearts were rapidly harvested, the atria, right ventricles, and major vessels were removed from the hearts, and the left ventricles were used for histological and biochemical studies. A group of mice underwent this surgical procedure with the exception of LAD occlusion and reperfusion and served as a sham control group.
Histopathological analysis
Tissue were fixed in 4% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin for routine histological evaluation.
Quantification of myocardial injury
Infarct size was determined by the triphenyl tetrazolium chloride-Evan’s blue technique (12)
. At the end of the reperfusion, the aorta was cannulated and 1% solution of triphenyl-tetrazolium chloride (TTC) was perfused into the aorta and coronary arteries. The heart was incubated at 37°C for 20 min. After incubation, the ligature around the left main coronary artery was retightened and 2 ml of 2% Evans blue dye was injected into the aorta to stain the area of the myocardium perfused by the patent coronary arteries. Thus, the area not at risk was determined by a blue staining and the area at risk was determined by negative staining. The atria, right ventricle, and major blood vessels were removed from the heart. The left ventricle was sliced into 1 mm-thick sections parallel to the atrioventricular groove and fixed in 10% formalin. The area at risk of infarction was colored brick red due to the formation of a precipitate resulting from the reaction of TTC with dehydrogenase enzymes. Loss of these enzymes from infarcted myocardium prevents formation of the precipitate; thus the infarcted area within the risk region remains pale yellow. Each slice was weighed and photographed under a dissective microscope. The pictures were scanned for calculation of the different areas (nonischemic, at risk, and infarcted) using the Adobe Photoshop program (12)
. The sizes of nonischemic area, area at risk, and infarcted area of each slice were calculated as a percentage of corresponding area multiplied by the weight of the slice.
Measurement of serum creatine phosphokinase activity
Serum levels of creatine phosphokinase were evaluated as an index of cardiac cellular damage by using a commercial kit (Sigma Chemical Co., St. Louis, MO).
Determination of apoptosis
Cell death by apoptosis was evaluated after measuring oligonucleosomal DNA fragments by a histochemical TdT ‘Tunel’-like staining (TdT-FragEL kit, Oncogene Research Products, Cambridge, MA) and by a DNA laddering assay. For in situ Tunel staining, frozen cardiac sections were permeabilized with protease K (2 mg/ml) in 10 mM Tris (pH 8) at room temperature for 20 min. Endogenous peroxidase was quenched with 3% H2O2 in methanol for 5 min. Sections were incubated with a reaction buffer composed of biotin-dCTP and of unlabeled dCTP and TdT enzyme (terminal deoxynucleotidyl transferase) in a humidified chamber at 37°C. In this assay, TdT binds to exposed 3'OH ends of DNA fragments and catalyzes the addition of biotin-labeled and unlabeled deoxynucleotides. Byotinilated nucleotides were detected using a streptavidin-horseradish peroxidase conjugate and diaminobenzidine (20)
. To quantitate the degree of apoptosis, apoptotic cells were counted by three independent observers blinded to the experimental protocol. The apoptotic index (number of myocardial nuclei labeled by the Tunel method/number of total myocardial nuclei) was calculated. For the DNA laddering assay, hearts were homogenized in a buffer containing 137 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 0.44 mM KH 2PO4, 0.34 mM Na2HPO4, 5.6 mM glucose, and 20 mM HEPES (pH 7.5). After centrifugation (1000 g, 10 min), pellets were solubilized at 4°C with lysis buffer (50 mM Tris; 10 mM EDTA; 0.5% Sarkosyl, pH 8.0) and digested in the presence of protease K (1 mg/ml) for 2 h at 50°C. Samples were treated with 20 µg RNase at 37°C for 1 h. Equal quantities of sample along with 1 kb DNA ladder (Life Technologies, Grand Island, NY) were subjected to electrophoresis on a 1.8% agarose gel containing ethidium bromide. DNA staining was visualized using the Bio-Rad gel documentation system (Bio-Rad Laboratories, Hercules, CA).
Northern blot analysis
Cardiac tissues were homogenized and total RNA was extracted by a modification of the thiocyanate-phenol-chloroform method of Chomczynski and Sacchi (21)
using the Trizol reagent (Life Technologies). RNA samples were further enriched for RNA by column spin using the Qiagen protocol (Qiagen, Valencia, CA). One-half of the eluted volume of RNA was electrophoresed on 1% formaldehyde agarose gel. For Northern blot analysis, the RNA was transferred to Magnacharge nylon membrane (Osmonic, Westborough, MA) in 20x SSC overnight by capillary action and cross-linked to the membrane with a short-wave UV cross-linker (Stratagene, La Jolla, CA). Transferred RNA was visualized by methylene blue staining. Membranes were prehybridized for 2 h at 42°C in NorthernMax solution (Ambion, Austin, TX) and hybridized overnight at 42°C with a murine iNOS cDNA probe (106 cpm/ml) labeled with [32P]dCTP. The blots were serially washed at 42°C using 2x sodium citrate, sodium chloride-0.1% sodium dodecyl sulfate (SDS) for 30 min, 1x sodium citrate, sodium chloride-0.1% SDS for 30 min, and at 55°C with 0.2x sodium citrate, sodium chloride-0.1% SDS for 30 min. After probing for iNOS, membranes were stripped with boiling 5 mM EDTA and rehybridized with a 32P-radiolabeled oligonucleotide probe for 18S ribosomal RNA as a housekeeping gene. The relative amount of mRNAs was evaluated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Expression of iNOS was normalized to 18S ribosomal RNA for comparative purposes. Densitometric analysis was performed using ImageQuant (Molecular Dynamics).
Measurement of nitric oxide synthase activity
Calcium-dependent and -independent conversion of L-arginine to L-citrulline in homogenates of hearts obtained from mice subjected to ischemia and reperfusion served as an indicator of ecNOS and iNOS activity, respectively (22)
. Hearts were homogenized on ice in a buffer composed of 50 mM Tris-HCl, 0.1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (pH 7.4). Conversion of [3H]-L-citrulline was measured in the homogenates as described. Homogenates (30 µl) were incubated in the presence of [3H]-L-arginine (10 µM, 5 kBq/tube), NADPH (1 mM), calmodulin (30 nM), tetrahydrobiopterin (5 µM), and EGTA (2 mM) for 20 min at 22°C. Reactions were stopped by dilution with 0.5 ml of ice-cold HEPES buffer (pH 5.5) containing EGTA (2 mM) and EDTA (2 mM). Reaction mixtures were applied to Dowex 50W (Na+ form) columns and the eluted [3H]-L-citrulline activity was measured by a Wallac scintillation counter (Wallac, Gaithersburg, MD).
Measurement of plasma nitrite/nitrate concentration
Nitrite/nitrate production, an indicator of NO synthesis, was measured in plasma samples as described previously (23)
. Nitrate in the plasma was reduced to nitrite by incubation with nitrate reductase (670 mU/ml) and NADPH (160 mM) at room temperature for 3 h. After 3 h, nitrite concentration in the samples was measured by the Griess reaction by adding 100 µl of Griess reagent (0.1% naphthalethylenediamine dihydrochloride in H2O and 1% sulfanilamide in 5% concentrated H3PO4; vol 1:1) to 100 µl samples. The optical density at 550 nm (OD550) was measured using a Spectramax 250 microplate reader (Molecular Devices, Palo Alto, CA). Nitrate concentrations were calculated by comparison with OD550 of standard solutions of sodium nitrate prepared in saline solution.
Immunohistochemistry for nitrotyrosine
Tyrosine nitration, a marker of nitrosative damage, was detected in cardiac sections by immunohistochemistry (19)
. Frozen sections (5 µm) were treated with 0.3% hydrogen peroxide for 15 min to block endogenous peroxidase activity and rinsed briefly in PBS. Nonspecific binding was blocked by incubating the slides with a blocking solution (0.1 M PBS containing 0.1% Triton X-100 and 2% normal goat serum) for 2 h. To detect nitrotyrosine, rabbit polyclonal anti-nitrotyrosine antibody was applied at 4°C overnight. Control sections included buffer alone or nonspecific purified rabbit IgG. Immunoreactivity was detected with a biotinylated goat anti-rabbit secondary antibody and the avidin-biotin-peroxidase complex (Vectastain Elite ABC kit, Vector Laboratories). Color was developed using diaminobenzidine.
Measurement of myeloperoxidase activity
Myeloperoxidase activity was determined as an index of neutrophil accumulation (19)
. Hearts were homogenized in a solution containing 0.5% hexa-decyl-trimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000 g at 4°C. An aliquot of the supernatant was allowed to react with a solution of tetra-methyl-benzidine (1.6 mM) and 0.1 mM H2O2. The rate of change in absorbance was measured by spectrophotometry at 650 nm. Myeloperoxidase activity was defined as the quantity of enzyme degrading 1 µmol of hydrogen peroxide/min at 37°C and expressed in units per 100 mg tissue.
Measurement of plasma levels of cytokines
Plasma levels of TNF-, IL-6, and IL-10 were evaluated by commercially available solid-phase sandwich ELISA kits (R&D Systems, Minneapolis, MN), using the protocol recommended by the manufacturer.
Subcellular fractionation and nuclear protein extraction
Tissue samples were homogenized with a Polytron homogenizer in a buffer containing 0.32 M sucrose, 10 mM tris-HCl, pH 7.4, 1 mM EGTA, 2 mM EDTA, 5 mM NaN3, 10 mM ß-mercaptoethanol, 20 µM leupeptin, 0.15 µM pepstatin A, 0.2 mM phenyl-methane-sulfonyl fluoride, 50 mM NaF, 1 mM sodium orthovanadate, 0.4 nM microcystin. The homogenates were centrifuged (1000 g, 10 min) and the supernatant (cytosol+membrane extract) was collected for evaluation of IB and HSP70 content and IKK activity as described below. The pellets were solubilized in Triton buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EGTA, 1 mM EDTA, 0.2 mM sodium orthovanadate, 20 µM leupeptin A, 0.2 mM phenyl-methane-sulfonyl fluoride). The lysates were centrifuged (15,000 g, 30 min, 4°C) and the supernatant (nuclear extract) was collected to evaluate JNK activity and DNA binding of NF-B and AP-1.
Western blot analysis of HSP70 and IB
Cytosol and nuclear content of HSP70 and cytosol degradation of IB were determined by immunoblot analyses. Cytosol and nuclear extracts were boiled in equal volumes of loading buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol) and 50 µg of protein loaded per lane on an 8–16% Tris-glycine gradient gel. Proteins were separated electrophoretically and transferred to nitrocellulose membranes. For immunoblotting, membranes were blocked with 5% nonfat dried milk in Tris-buffered saline (TBS) for 1 h and incubated with primary antibodies against HSP70 or IB for 1 h. Membranes were washed in TBS with 0.1% Tween 20 and incubated with secondary peroxidase-conjugated antibody; the immunoreaction was visualized by chemiluminescence. Densitometric analysis of blots was performed using ImageQuant (Molecular Dynamics).
Assay of IKK and JNK activity
Activity of IKK and JNK was determined by immune complex kinase assay and estimated as the ability to phosphorylate glutathione-S-transferase (GST)-IB or GST-c-Jun, respectively (24)
. After immunoprecipitation of lysate with specific antibody directed to IKK
or JNK1, the immunoprecipitate was incubated for 30 min at 30°C in 40 µl of reaction buffer (25 mM HEPES, pH 7.6, 20 mM MgCl2, 20 mM glycerolphosphate, 0.1 mM sodium orthovanadate, 2 mM dithiothreitol, 25 µM ATP, and 5 µCi of [-32P]ATP. GST-IB (1–54) (1 µg) was used as substrates for the IKK complex; GST-c-Jun (1–79) (1 µg) was used as substrate for JNK. Reaction products were separated by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. Densitometric analysis was performed using ImageQuant (Molecular Dynamics).
Electrophoretic mobility shift assay
Electrophoretic mobility shift assays (EMSAs) were performed as described (25)
. Oligonucleotide probes corresponding to NF-B consensus sequence (5'-AGT TGA GGG GAC TTT CCC AGG C-3') or AP-1 consensus sequence (5'-CGC TTG ATG ACT CAG CCG GAA-3') were labeled with -[32P]ATP using T4 polynucleotide kinase and purified in Bio-Spin chromatography columns (Bio-Rad). Ten micrograms of nuclear protein were preincubated with EMSA buffer (12 mM HEPES pH 7.9, 4 mM Tris-HCl pH 7.9, 25 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1 mM DTT, 50 ng/ml poly [d(I-C)], 12% glycerol v/v, and 0.2 mM PMSF) on ice for 10 min before addition of the radiolabeled oligonucleotide for an additional 10 min. Protein–nucleic acid complexes were resolved using a nondenaturing polyacrylamide gel consisting of 5% acrylamide (29:1 ratio of acrylamide:bisacrylamide) and run in 0.5x TBE (45 mM Tris-HCl, 45 mM boric acid, 1 mM EDTA) for 1 h at constant current (30 mA). Gels were transferred to Whatman 3M paper, dried under a vacuum at 80°C for 1 h, and exposed to photographic film at -70°C with an intensifying screen. Densitometric analysis was performed using ImageQuant (Molecular Dynamics).
Materials
Primary anti-nitrotyrosine antibody was purchased from Upstate Biotech (Saranac Lake, NY). The primary antibodies directed at IB, HSP70, JNK1, and IKK and the oligonucleotides for NF-B and AP-1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Reagents, secondary, and nonspecific IgG antibodies for immunohistochemical analysis were from Vector Laboratories (Burlingame, CA). The ELISA kits for TNF-, IL-6, and IL-10 were obtained from R&D Systems (Minneapolis, MN). All other chemicals were from Sigma/Aldrich (St. Louis).
Data analysis
All values in the figures and text are expressed as mean ± SE of n observations (n=6–12 animals for each group). The results were examined by analysis of variance, followed by the Bonferroni’s correction post hoc t test. A P value of less than 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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In wild-type iNOS+/+ mice, occlusion (60 min) of the LAD followed by reperfusion (60 min) resulted in a mild myocardial injury (see Fig. 1
for histology and Fig. 2
for infarct staining). The infarcted area corresponded to 38.0 ± 2.3% of the area at risk and was associated with elevated serum levels of creatine phosphokinase activity (Fig. 3
A). The absence of a functional iNOS gene in iNOS-/- mice resulted in a significant augmentation of reperfusion injury of previously ischemic myocardium. The histological features were characterized by a widespread disruption of the myocardium with a massive presence of contraction bands (Fig. 1)
. The infarcted area was increased significantly compared with iNOS+/+ littermates (P<0.05) and corresponded to 53.7 ± 2.3% of the area at risk. Areas at risk were similar in iNOS+/+ and iNOS-/- mice (41.6±3.2% and 47.5±3.0% of total left ventricle, respectively). In iNOS-/- mice, serum levels of creatine phosphokinase activity were similar to those of iNOS+/+ mice (Fig. 3A
).
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Absence of iNOS increases cellular death by apoptosis
To test whether disruption of myocardial architecture was associated with cell death by apoptosis, we next measured oligonucleosomal DNA fragmentation. In gel electrophoresis of DNA revealed a significant fragmentation in hearts of iNOS-/- mice 60 min after reperfusion (Fig. 3B
), confirmed by in situ Tunel staining. As shown in Fig. 4
, myocardial ischemia (60 min) followed by reperfusion (60 min) resulted in the marked appearance of dark brown apoptotic cells scattered throughout the cell population in the infarcted area in tissues from iNOS-/- mice. On the contrary, only a small number of cells were stained dark brown in the left ventricle of wild-type mice, indicating a significant decrease in apoptotic cell death (Fig. 4)
. Apoptotic indices of hearts from iNOS+/+ mice vs. iNOS-/- mice were 0.19 ± 0.05% vs. 0.86 ± 0.11%, respectively (P<0.05). No apoptotic cells were observed in sections from sham iNOS+/+ and iNOS-/- mice.
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Production of NO and formation of nitrotyrosine are abolished in the absence of iNOS
To determine whether myocardial damage was associated with changes in NO release, we evaluated iNOS expression, enzyme activity in heart samples, and plasma levels of NO stable metabolites, nitrate, and nitrite. According to Northern blot analysis, expression of iNOS mRNA increased in a time-dependent fashion after reperfusion in iNOS+/+ animals (Fig. 5
) but was absent in iNOS-/- animals (data not shown). Changes in ecNOS activity were similar in iNOS-/- and wild-type mice. At the end of reperfusion, catalytic activity of ecNOS increased significantly in both iNOS-/- and wild-type mice (3.23±0.26 and 3.87±0.4 pmol/min/mg, respectively) when compared with preischemic activity (2.58±0.32 and 2.99±0.07 pmol/min/mg, respectively; P<0.05). During the reperfusion period, however, a sharp increase in iNOS catalytic function was observed in iNOS+/+ mice. These events were associated with a significant increase of plasma nitrate/nitrite in iNOS+/+ mice only (Fig. 6
).
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The elevation of iNOS activity and increase in NO production correlated with the appearance of a positive immunohistochemical staining for nitrotyrosine, which was scarce and localized mainly in the injured endocardium of the peri-infarction zone in hearts of iNOS+/+ mice. In contrast, nitrotyrosine staining was virtually abolished in the iNOS-/- mice after reperfusion (Fig. 7
).
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Neutrophil infiltration is increased in the absence of iNOS
A hallmark of reperfusion injury is the accumulation into the injured tissue of neutrophils, which augments the damage to vascular and parenchyma cellular elements. Therefore, we next evaluated the extent of neutrophil infiltration in myocardial tissue by measuring the activity of myeloperoxidase, an enzyme specific to granulocyte lysosomes and therefore directly correlated with the number of neutrophils. Myeloperoxidase activity was slightly elevated at the end of the reperfusion period in iNOS+/+ mice. In contrast, in iNOS-/- mice, tissue myeloperoxidase activity increased markedly as early as 15 min after reperfusion and was significantly higher than myeloperoxidase activity of iNOS+/+ animals (Fig. 8
).
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Absence of iNOS alters production of TNF-, IL-6, and IL-10
A substantial increase in TNF-, IL-6, and IL-10 production was found in iNOS+/+ mice after myocardial ischemia and reperfusion. TNF- and IL-10 levels were elevated soon after reperfusion and tended to be stable until the end of the experimental period. IL-6 levels reached a peak 60 min after reperfusion. In iNOS-/- mice, TNF- and IL-6 production exhibited different kinetics, since levels were increased significantly during the early reperfusion vs. those of iNOS+/+ animals and tended to decrease later in the reperfusion period. In contrast, IL-10 production was significantly reduced during the early reperfusion compared with iNOS+/+ animals and tended to increase later in the reperfusion period (Fig. 9
).
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HSP70 is decreased in the absence of iNOS
To establish the mechanisms inducing myocardial cell death, we next determined the myocardial expression of HSP70, a cardioprotective protein with putative anti-apoptotic effects (26
, 27)
. Western blot analyses indicated that HSP70 expression was increased by ischemia and reperfusion injury in both cytosol and nuclear compartments in iNOS+/+ animals. In contrast, no significant increase above basal levels was observed in ischemic or reperfusion conditions in cytosolic extracts of iNOS-/- animals. However, densitometric analysis revealed that in iNOS-/- mice, nuclear content of HSP70 was significantly depressed when compared with the nuclear content of HSP70 in wild-type mice (Fig. 10
).
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Activation of NF-B, degradation of IB and activity IKK are depressed in the absence of iNOS
To investigate the cellular mechanisms by which iNOS-derived NO may attenuate reperfusion-induced injury, we evaluated the nuclear activation of NF-B, a major transcription factor involved in the signal transduction of the reperfusion injury (5
6
7)
. Reperfusion after myocardial ischemia in iNOS+/+ mice resulted in the early activation of NF-B, with activity reaching a maximum after 15 min of reperfusion and declining thereafter. In contrast, in iNOS-/- mice, DNA binding activity of NF-B was initially depressed earlier in reperfusion but increased 45 and 60 min after reperfusion (Fig. 11
A, B). At later stages of reperfusion, NF-B activation was similar in both experimental groups. In iNOS+/+ mice, the fold increase in NF-B activity was 1.39 ± 0.10 and 1.15 ± 0.12 vs. basal levels at time 0 (set to 1), respectively, 2 and 4 h after reperfusion. In iNOS-/- mice, the fold increase in NF-B activity was 1.32 ± 0.18 and 1.05 ± 0.13 vs. basal levels at time 0 (set to 1), respectively, 2 and 4 h after reperfusion.
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Degradation of IB by a phosphorylation- and ubiquitination-dependent pathway represents an important event for the unmasking of NF-B translocation sequences and the initiation of transcription by the nuclear factor (7)
. To provide more insight into the regulatory role of iNOS, we determined the reperfusion-induced degradation of IB and activity of IKK. We found that in iNOS+/+ mice, IB content was partially degraded 0 min after ischemia and 15 min after reperfusion, but was replenished later after reperfusion (Fig. 12
). These events were preceded by an increase in the phosphorylative activity of IKK. Enzyme activity rose 30 min after ischemia, reached a peak 30 min after reperfusion, and declined thereafter (Fig. 11C, D
). In contrast, in iNOS-/- mice IB content increased after reperfusion. The increase of IKK activity above basal levels was delayed 30 min after reperfusion and was significantly reduced compared with iNOS+/+ mice (Figs. 11
, 12)
.
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Activation of AP-1 and JNK activity are reduced in the absence of iNOS
As activation of AP-1 has been implicated in myocardial reperfusion injury, we further determined the nuclear activation of this factor (4
, 28)
. In iNOS+/+ mice, DNA binding activity of AP-1 steadily increased after reperfusion. In iNOS-/- mice, activity of AP-1 exhibited similar kinetics, increasing in a time-dependent fashion after reperfusion. However, the degree of activation was significantly reduced when compared with wild-type mice (Fig. 13
A, B). Since phosphorylation of components of AP-1 such as c-Jun by JNK represents an important event for the stability and activation of the transcription factor AP-1 (4
, 24)
, we further determined the nuclear activity of JNK. A time course study showed that JNK activity exhibited similar kinetics in both iNOS-/- and wild-type mice: the enzymatic activity increased as early as 15 min after reperfusion and declined thereafter. However, in iNOS-/- mice, the degree of activity of JNK was significantly reduced compared with wild-type controls (Fig. 13C, D
).
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At later stages of reperfusion, AP-1 activation was maintained at a higher, but not statistically significant, degree in iNOS-/- mice vs. iNOS+/+ mice. In iNOS+/+ mice, the fold increase in AP-1 activity was 2.24 ± 0.21 and 1.65 ± 0.52 vs. basal levels at time 0 (set to 1), respectively, 2 and 4 h after reperfusion. In iNOS-/- mice, the fold increase in AP-1 activity was 2.81 ± 0.46 and 2.32 ± 0.16 vs. basal levels at time 0 (set to 1), respectively, 2 and 4 h after reperfusion.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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B and JNK/AP-1 signal transduction pathways are activated during myocardial ischemia and reperfusion and that their regulation may be subjected to the endogenous production of iNOS-derived NO.
NO is an endogenous cardioprotective factor
Despite extensive research, the role of NO in ischemia and reperfusion injury remains controversial and is yet to be defined. In our study, we demonstrated that in wild-type mice iNOS was expressed very early during reperfusion, resulting in increased catalytic activity and elevated production of NO. These events were associated with a reduced infarct size. As the catalytic activity of ecNOS was increased at the end of the reperfusion period to a similar degree in both iNOS-/- and iNOS+/+ mice, it is possible that iNOS-derived NO is responsible for the cardioprotection. Our findings agree with several reports demonstrating that NO functions as a protective agent during reperfusion injury. In isolated perfused Langendorff preparations, a rapid superinduction of iNOS during early reperfusion attenuated a hyperdynamic response (11)
. Approaches to remove NO by pharmacological or genetic inhibition of iNOS have also been shown to exacerbate reperfusion injury in the heart (12
13
14
15)
, whereas approaches to deliver NO by donors have been shown to ameliorate tissue damage (10
, 29
30
31
32)
. However, there is equally compelling evidence that NO and other reactive nitrogen species generated from iNOS, including peroxynitrite, may also contribute to postischemic myocardial injury (33
, 34)
. We have previously demonstrated that peroxynitrite is formed during reperfusion and induces structural and functional alteration of cardiomyocytes (19
, 35)
. Recent evidence indicates that several chemical reactions involving nitrite, peroxynitrite, hypochlorous acid, and peroxidases can induce tyrosine nitration and may contribute to tissue damage (36)
. In this current study, we found that formation of nitrotyrosine occurred rarely during reperfusion and was mainly confined only in the peri-infarction zone of the endocardium site in wild-type mice. Although nitrotyrosine formation was virtually abolished in iNOS-/- mice, its abrogation did not correlate with an amelioration of the severity of injury. Our data suggest that formation of highly toxic nitrating agents may not be considered the sole mechanism of oxidant injury during early reperfusion and that their inhibition is not sufficient to limit myocardial damage.
In support of this hypothesis, we found that leukocyte infiltration at a lesser degree in wild-type mice correlated well with the moderation of postreperfusion tissue damage. This finding suggests that other reactive species derived form neutrophils contribute to myocardial reperfusion injury and that NO may also provide protection against reperfusion injury by preventing neutrophil infiltration and the subsequent release of oxidants. It is also possible that in the pathological setting of myocardial injury, the biological effects of NO are influenced by the cellular redox state. It has been demonstrated that although NO markedly attenuates postischemic myocardial tissue damage, its one-electron reduction product nitroxyl (NO-) exerts completely opposite effects and aggravates myocardial reperfusion injury (37)
. Furthermore, it has been proposed that peroxynitrite at low concentrations may attenuate contractile dysfunction and may exert significant cardioprotective and vasculoprotective effects in myocardial ischemia and reperfusion (38)
. Therefore, it appears that different experimental conditions can affect the molecular reactivity of NO. These variables may include differences in rate, timing of NO release, extension of tissue damage, and consequently the cellular environment of target molecules.
Induction of NO synthesis reduces cell death by apoptosis
After ischemia and reperfusion of the myocardium, cardiomyocytes die via necrosis and apoptosis. In contrast to the swelling and membrane rupture associated with necrosis, apoptotic cells shrink and maintain their membrane integrity (3
, 39
40
41)
. We found that the genetic absence of iNOS resulted in increased apoptosis, whereas activation of the inducible enzyme in wild-type mice resulted in a much-decreased apoptosis in cardiomyocytes that correlated with reduction of the infarction zone. Surprisingly, serum levels of creatine phosphokinase were similarly elevated in both iNOS+/+ and iNOS-/- mice. It is noteworthy that serum creatine phosphokinase is considered a standard biomarker for cell necrosis as the enzyme is released from the cytosol due to damage of the plasma membrane of dead myocytes (42)
. It is conceivable that elevated serum levels of creatine phosphokinase do not correlate with the apoptotic cell death, as apoptotic cells, which maintain an intact membrane, may retain the enzyme. Therefore, our findings suggest that the cardioprotective effects of early production of NO may be attributable primarily to its anti-apoptotic action on cardiomyocytes and necrotic death is not affected. Our data suggest that apoptosis rather than necrosis is a major contributor to infarct size during early reperfusion. We found that expression of the cardioprotective HSP70 was enhanced in wild-type animals but depressed in mice with genetic disruption of iNOS. The major changes in HSP70 expression were observed primarily in the nuclear compartment, i.e., where chromatin-condensation and DNA fragmentation take place during apoptotic death. Therefore, although the role of other anti-apoptotic proteins cannot be ruled out, our data suggest that iNOS-derived NO is a requisite for the rapid induction of HSP70 expression in cardiomyocytes. Our results agree with previous findings demonstrating that HSP70 has a critical role in anti-apoptosis, counteracting apoptotic signals in a variety of cells (27)
. Exogenous NO or preinduction of iNOS has been shown to stimulate expression of the inducible HSP70, subsequently preventing hepatocytes from TNF-- and actinomycin D-induced apoptosis (43)
.
It is also interesting that the increase in cardiac damage and apoptosis cell death in the absence of a functional iNOS gene was associated with alteration of the kinetics of cytokine production. Production of TNF- and IL-6, which have been implicated in promoting myocyte death and heart failure disease progression after infarction (44)
, was increased significantly at earlier time points of reperfusion in iNOS-/- mice compared with lower levels of wild-type animals. In contrast, production of the anti-inflammatory IL-10, which has been suggested to afford protection during ischemia and reperfusion injury (25
, 45)
, was decreased significantly earlier in reperfusion with iNOS-/- mice vs. higher levels in wild-type animals. Nossuli and collaborators (46)
have proposed that the acute surgical trauma may increase background of cytokine induction, thus causing great variability and confusing the interpretation of the data. In a model of open chest surgery, the authors found that mRNA expression of IL-6 and TNF- increased 3 h after the surgery. In our model, which is similar to the one adopted by Nossuli, we found detectable levels of cytokines soon after the surgery (i.e., at the end of the ischemia, 1 h after the open chest surgery). Furthermore, differences in the kinetics of cytokine release paralleled the differences in infarct size and apoptosis between the iNOS+/+ and iNOS-/- groups. It therefore appears that in our model of myocardial injury, the increase in cytokine levels may be attributed to the ischemic injury rather than to surgical manipulation only. Taken together, our data suggest that iNOS-derived NO is crucial to maintain host defense against reperfusion damage by promoting an anti-inflammatory response in the early stage of reperfusion.
Genetic absence of iNOS alters signal transduction mediated by NF-B and AP-1
Several cellular mechanisms, including the mode of gene regulation and signal transduction, may account for the role of NO in the modulation of myocardial reperfusion injury. Numerous experimental studies have proved that activation of both NF-B and AP-1 are implicated in myocardial reperfusion injury. Enhancement of AP-1 and NF-B DNA binding activity has been found in areas of infarction in rats subjected to myocardial ischemia and reperfusion (28
, 47)
. A similar activation of NF-B and AP-1 occurs during reperfusion in other previously ischemic organs and tissues, such as brain and liver (48
, 49)
. Of particular clinical relevance, nuclear translocation of both NF-B and AP-1 has been found in cardiac biopsies of patients with unstable angina (50)
. These findings strongly suggest that NF-B and AP-1 can work cooperatively to produce a specific inflammatory response.
Under physiological conditions, NF-B is sequestered in an inactive form in the cytosol through noncovalent interactions with inhibitor proteins such as IB. Phosphorylation of IB at serine residues triggers the multi-ubiquitination of IB and its subsequent degradation by the 26S proteasome (5
6
7)
. The protein kinase that phosphorylates IB has been identified as a complex named the IB complex kinase (IKK) and is composed of at least two catalytic subunits, IKK and IKKß, and the regulatory subunit IKK (7
, 51)
. After dissociation from its inhibitor, NF-B translocates to the nucleus, where it leads to the activation of various anti- and proinflammatory mediators (5
6
7)
. In our study, we found that in iNOS+/+ mice activation of NF-B was increased markedly as early as 15 min after reperfusion. In contrast, in the absence of a functional gene for iNOS, activity of NF-B was depressed earlier in reperfusion and increased only at the end of reperfusion. Under the experimental conditions used in our laboratory, the impairment and delay of NF-B activity observed in iNOS-/- mice appear to be sequential events of depressed and delayed IKK activity. To our knowledge, this is the first report demonstrating that iNOS-derived NO is a requisite for a rapid activation of the phosphorylative activity of IKK. We found that IB content was potentiated even in the absence of iNOS. This suggests that NO may regulate stability and content of IB through other cellular mechanisms in addition to the modulation of IKK-mediated phosphorylation and degradation. The present study is in accord with reports that ascribe to NO a regulatory role on gene expression and NF-B activity. It has been demonstrated that at lower concentrations, NO potentiates endotoxin-induced NF-B activity but at higher concentrations exerts an inhibitory effect in in vitro murine macrophages (52)
.
Our results suggest that the early activation of NF-B in wild-type animals may reflect a defensive reaction and participate in cardioprotection against myocardial infarction. In support of our hypothesis, several in vitro studies have suggested that NF-B plays a role as a survival factor, responsible in part for ‘turning on’ genes that could block cell death by apoptosis (51
, 53)
. In in vitro myocytes, for example, apoptosis was increased markedly in the absence of NF-B activation (54)
. Therefore, it is conceivable that a complete inhibition of NF-B activation, especially during early reperfusion, may enhance reperfusion-induced cytotoxicity. However, it must be considered that NF-B is also a prominent transcriptional promoter of many proinflammatory cytokines such as the IL-1, IL-2, IL-6, IL-8, TNF-, interferon , and cell adhesion proteins, including intercellular adhesion molecule 1 and vascular adhesion molecule (5
6
7)
. Our data contrast with the recent hypothesis that inhibition of NF-B activation may exert protective effects in ischemia and reperfusion injury.
Previous reports have described that in vivo transfer of the cis element ‘decoy’ against the NF-B binding site reduced the extent of myocardial infarction in rats (55)
. We have also demonstrated that pharmacological inhibition of IKK activity and NF-B reduces infarct size, neutrophil infiltration, and oxidative damage in a rat model of myocardial reperfusion injury (56)
. Nevertheless, according to our present data, it appears that the initial and temporal activity of NF-B may be a necessary pathway for the early anti-inflammatory response. In support of this, we found that at later stages of reperfusion NF-B was in fact similarly activated in wild-type and iNOS-/- animals.
Nuclear activation of the transcription factor AP-1 during reperfusion in the ischemic myocardial tissue of iNOS-/- mice was reduced, but not completely depressed as NF-B was, and exhibited kinetics similar to wild-type mice, increasing in a time-dependent fashion during the early reperfusion. Later in reperfusion, however, AP-1 activation was maintained to a higher (but not statistically significant) degree in iNOS-/- vs. wild-type mice. Examined at a more molecular level, the earlier reduction of AP-1 activity observed in iNOS-/- mice appears to be a sequential event of reduced JNK nuclear activity. In fact, it has been proposed that JNK translocates to the nucleus during ischemia of perfused rat hearts and then is activated during reperfusion (24)
. JNK activation is an important signal transduction for activation of the nuclear transcription factor AP-1 through phosphorylation of the subcomponent c-Jun (7)
. This suggests that endogenous NO may also target the AP-1 signaling pathway through modulation of the phosphorylative activity of JNK.
The JNK/AP-1 pathway has been observed in response to growth factors, cytokines, ischemia, and stress signals and has been implicated in cellular dysfunction and apoptosis of several cell types (24
, 57
58
59)
. Similar to NF-B pathway, AP-1 has also been implicated in regulated gene transcription of endothelial adhesion molecules and cytokines (60
61
62)
. Therefore, we can speculate that the ability to release proinflammatory cytokines such as IL-6 and TNF- in iNOS-/- mice, which exhibited a repressed NF-B pathway, may be due at least in part to maintenance of the JNK/AP-1 pathway throughout the early and late stages of reperfusion. Nevertheless, we cannot exclude the involvement of other signaling pathways, which will have to be further investigated.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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and IL-6 and the positive modulation of secretion of the anti-inflammatory mediators IL-10 and HSP70 through early activation of gene transcription. Differences in the rate and timing of NO release, as well as the species and other experimental conditions, may account for the reported controversial and different effects of NO. Nevertheless, it appears that during the very early stage of reperfusion within the heart, generation of free radicals induces early gene expression of iNOS. The temporal and spatial restriction of iNOS activation may serve to focus NO generation in an appropriate defense response against inflammation. This early defense response appears to be regulated by both NF-B and AP-1 signaling pathways (Fig. 14
).
|
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ACKNOWLEDGMENTS |
|---|
Received for publication July 17, 2001.
Revision received November 28, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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B-NF-B structures: at the interface of inflammation control. Cell 11,729-731
B on cardiomyocyte apoptosis during myocardial ischemic stress adaptation. FEBS Lett 443,331-336[CrossRef][Medline]
B kinase (I 75 K) and NF-B: key elements of proinflammatory signalling. Semin. Immunol. 12,85-98[CrossRef][Medline]
