Cardiac-Specific Overexpression of Inducible Nitric Oxide Synthase Does Not Result in Severe Cardiac Dysfunction
Nitric oxide (NO), a potent regulator of myocardial contractility, has been implicated in the development of heart failure; however, no study exists describing the relation between expression of inducible nitric oxide synthase (iNOS), formation of NO in vivo, and cardiac contractility. We have therefore generated transgenic (TG) mice overexpressing iNOS under the cardiospecific α-myosin heavy chain (α-MHC) promoter. In vitro, iNOS activity in hearts of two transgenic lines was 260- to 400-fold above controls (wild type [WT]), but TG mice were viable and appeared normal. Ventricular mass/body weight ratio did not differ; heart rate and cardiac output as well as mean arterial blood pressure were decreased by 10%. NOx levels of hearts and blood of TG mice were 2.5- and 2-fold above WT controls, respectively. In the isolated heart, release of the NO oxidation products nitrate and nitrite, an index of in vivo NOS activity, was 40-fold over WT. However, cardiac hemodynamics and levels of ATP and phosphocreatine were unaltered. The high iNOS activity was associated with reduced cardiac l-arginine in TG hearts to only 15% of the WT, indicating limited substrate availability, whereas l-citrulline was 20-fold elevated. Our findings demonstrate that the heart can tolerate high levels of iNOS activity without detrimental functional consequences. The concept that iNOS-derived NO is the triggering factor in the pathomechanism leading to heart failure therefore needs to be reevaluated.
It is well established that nitric oxide (NO) exhibits potent cardiovascular actions that include, aside from its vasodilatory effect, the inhibition of contractile force development1 most likely by modulation of mitochondrial respiration.2 NO inhibits the positive inotropic response to β-adrenergic stimulation in patients with left ventricular dysfunction3 and in patients with severe heart failure.4 Heterogeneous basal expression of nitric oxide synthase (NOS) isoenzymes was recently reported showing a significant gradient of endothelial NOS (eNOS) across the left ventricular wall and detectable neuronal NOS (nNOS) and basal inducible NOS (iNOS) in cardiomyocytes with expression of both being highest in left ventricular endocardial myocytes.5 NO as an endogenous regulator of myocardial contractility has received considerable attention, and the progress is summarized in several recent reviews.6–10⇓⇓⇓⇓
The notion of the potent cardiodepressant activity of NO raised the possibility that cardiac synthesis of NO may modulate cardiac contraction in disease states such as the failing heart. Advanced stages of chronic heart failure are often associated with systemic and cardiac cytokine activation,11,12⇓ and cytokines are well known to be potent stimulators of the expression of iNOS also in cardiomyocytes.13 de Belder et al14 were the first to show enhanced activities of iNOS in right ventricular tissue from patients with dilated cardiomyopathy (DCM). Later it was shown that iNOS activity can be measured in myocarditis, ischemic heart disease (IHD), and valvular heart disease (VHD),15 although negative findings for IHD and VHD were also reported.16 Overexpression of iNOS was also found in human cardiac allografts,17,18⇓ and the expression levels were found to correlate with ventricular contractile dysfunction. More recently significant expression of iNOS in the human heart was shown to be associated with a pronounced reduction of eNOS.4,5,19⇓⇓ Together, the experimental and clinical results led to the concept that an enhanced production of NO by iNOS is causally related to the observed contractile dysfunction in heart failure.
Studies performed so far could not answer the question whether iNOS expression is crucial in the development of heart failure or whether iNOS is just a cofactor in the context of a more complex cytokine-mediated process. In addition, there are no data on the rate of endogenous NO production necessary to elicit cardiac dysfunction by measuring iNOS expression both at the mRNA and protein levels and the relation of these parameters to in vivo NO formation and contractility. We have therefore overexpressed iNOS under the cardiospecific α-myosin heavy chain (α-MHC) promoter in transgenic mice, thus allowing the assessment of the role of iNOS without changes in cytokine levels. Quite unexpectedly, we found no signs of heart failure despite massive overexpression of iNOS. This has led us to uncover mechanisms by which the heart in vivo can functionally deal with pathological NO production, which includes limitation of endogenous supply of l-arginine and rapid intracellular metabolism.
Materials and Methods
Production of Transgenic Mice
Transgenic mice were generated by pronuclear microinjection of linearized transgene (1 ng/μL) in fertilized eggs of superovulated FVB/N (Tierversuchsanlage, Düsseldorf, Germany) and reimplantation into 0.5-day postcoital pseudopregnant C57Bl6/CBA F1 hybrid foster mothers (M & B, Denmark). All procedures were performed using standard techniques,20 and all animals were treated according to National Institutes of Health guidelines.
PCR and Southern Analysis
Founder mice harboring the transgene were identified by PCR and subsequent Southern blot analysis of genomic DNA isolated from tail biopsies.
Total RNA isolated from transgenic and control hearts was prepared by the guanidinium thiocyanate-phenol-chloroform extraction procedure.21 After electrophoresis in formaldehyde-containing agarose gel, RNA was transferred onto nylon membranes. Hybridization was performed using 32P-labeled whole iNOS cDNA (3.5 kb) as probes and exposure to film.
Protein extracts were prepared by homogenization of hearts in 50 mmol/L Tris (pH 7.5), 1 mmol/L EDTA, and 1 mmol/L PMSF. iNOS was detected by monoclonal antibodies directed against mouse iNOS (Transduction Laboratories) according to the manufacturer’s instructions. Protein bands were detected by horseradish peroxidase-labeled goat anti-mouse antibodies (Sigma) by use of the ECL detection system (Amersham).
For immunohistochemistry, organs were rapidly excised and frozen in OTC reagent (Miles). To make results comparable, wild-type (WT) and transgenic (TG) hearts were mounted onto the same carrier and slices were processed simultaneously on the same slides. Sections (10 μm) were cut on a cryostat and processed as described previously.22 For immunostaining, sections were incubated with primary polyclonal rabbit anti-iNOS antibodies (UBI) diluted 1:2000 with TBS/1% normal goat serum for 24 hours at 4°C. Binding of primary antibody was visualized with a VectaStain anti-rabbit ABC kit (Vector Laboratories) using 3′-3′-diaminobenzidine as a chromogene. Trichrome staining was performed according to Masson.
RT-PCR was performed in an automated thermal cycler and detected with the GeneAmp 5700 sequence detection system (PE Biosystems) using SYBR Green fluorescence for quantification.
Measurement of iNOS activity in hearts extracts in vitro by l-citrulline formation was performed by a reported method23 using 200 μmol/L l-arginine and [3H]l-arginine (4×105 cpm/sample) for 15 minutes at 37°C. Specific iNOS activity was calculated as the S-ethylisothiourea (ETU)–sensitive formation of [3H]l-citrulline · min−1 · mg−1 of protein.
Argininosuccinate Synthetase (ASS) Assay
Total hearts were homogenized in 500 μL of buffer containing 50 mmol/L Tris-HCl (pH 7.5) and 1 mmol/L PMSF. After incubation for 30 minutes on ice and centrifugation at 13 000 rpm for 30 minutes, supernatants were used for the ASS assay. ASS activity was assayed as described by O’Brien.24
Determination of Nitrite and Nitrate
Nitrate and nitrite accumulating in the coronary venous effluent were detected by chemoluminescence with an NO analyzer (NOA280, Sievers Inc). Nitrite was converted to NO by potassium iodide in acetic acid and NOx (NO3− and NO2−) was reduced to NO with VCl3 according to the manufacturer’s protocol. Owing to traces of nitrate contaminations in the perfusion medium (≈73 pmol/mL), basal nitrate release could not be determined because coronary venous levels were not above threshold detection.
Cardiac NOx levels were measured according to a modified protocol of Matsuoka et al.25
Preparation of heart extracts and measurements of NADPH were performed according to published protocols.26
For echocardiography (Agilent Sonos 5500), a 12-MHz pediatric-phased array transducer equipped with a homemade 20-mm standoff was used. The chest was shaved, and animals were held in the supine position in one hand of the investigator. Prewarmed ultrasound transmission gel was applied to the precordium. The heart was imaged in two-dimensional (2D) mode in the parasternal long-axis view and 2D-guided M-mode images were obtained at the level of the papillary muscles for measurement of wall thickness and chamber dimensions.
Blood Pressure Measurement
For measurement of blood pressure, mice weighing 21 to 33 g (10 to 32 weeks old) were anesthetized with urethane (1.5 g · kg−1) and placed on a controlled warming table (37°C). A stretched polyethylene catheter (inner diameter 0.4 mm) was filled with saline/heparin (100 U · mL−1) and inserted into the right carotid artery and connected to a Statham P23XL transducer. Blood pressure and heart rate were continuously recorded using a Mac Laboratory data acquisition system (ADI Instruments).
NMR Langendorff Setup
Preparation of murine hearts (6 TG, 6 WT) and retrograde perfusion at 100 mm Hg with modified Krebs-Henseleit buffer gassed at 95% O2/5% CO2 were performed essentially as described previously.27
Amino Acid Quantification
Separation and quantification of individual amino acids in serum and heart muscle were performed by single-column cation exchange chromatography.28
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
Molecular Characterization of Transgenic Mice
For cardiac-specific overexpression, the iNOS cDNA29 was placed under the control of the α-MHC promoter30 (Figure 1a), known to direct high levels of transgene expression to mouse ventricles with the onset of expression on day 1 after birth.31 Seventeen founders were used to establish transgenic lines by breeding with FVB mice.
Western blot analysis revealed that of the 17 lines, 10 showed iNOS expression with expression levels differing at least 200-fold among lines. For further analysis, two highly expressing lines (TG5, TG36) were chosen as shown in a representative Western blot in Figure 1c. Southern blot analysis revealed that the varying amounts of iNOS expression did not correlate with the copy number of transgenes integrated into the mouse genome (Figure 1b).
To assess tissue specificity of expression, Northern blot analysis of total RNA prepared from heart, liver, kidney, lung, spleen, and skeletal muscle was performed (Figure 1d). Strong iNOS expression could be detected in heart already after 1 day of exposure. To investigate whether low-level expression occurred in other organs, long-term autoradiography was performed. After 6 weeks, a faint iNOS expression was detected in lungs as well. RT-PCR with primers hybridizing to exon 2 of the α-MHC gene and the 5′ region of the iNOS cDNA yielded the same results (data not shown). The low-abundance expression in the lung reflects the normal expression of α-MHC in pulmonary vasculature.32
Immunohistochemistry demonstrated a homogeneous, cytosolic distribution of iNOS protein in cardiac myocytes (Figures 2a and 2b). Masson’s trichrome staining, discriminating between cytoplasm (red) and connective tissue (blue), revealed no differences between control and transgenic mice (Figures 2c and 2d). NOS activity in vitro as determined by the citrulline assay was found to be 697±238 (n=8) pmol · min−1 · mg−1 protein in TG5 and 442±224 pmol · min−1 · mg−1 protein in TG36 (n=6) mice compared with 1.7±3.1 pmol · min−1 · mg−1 protein in WT mice (n=6).
Functional and Metabolic Analysis of Heart Function
iNOS-overexpressing animals were healthy and without unusual mortality. They bred normally and produced litters of normal size and similar physical activity. Functional consequences of iNOS overexpression were studied in conscious mice by means of echocardiography (Figure 3). Studies were performed on WT, TG5, and TG36 mice and revealed a small but significant decrease in cardiac output in transgenic mouse hearts whereas shortening fraction was slightly reduced. Ventricular mass/body weight ratio and heart rate did not differ between WT and TG mice. In addition, we measured arterial blood pressure in anesthetized mice. As shown in Table 1, we found a small but significant decrease (−8 mm Hg) in mean and diastolic arterial blood pressure, whereas heart rate also was slightly decreased.
For further characterization of the cardiac phenotype, hemodynamic parameters such as left ventricular developed pressure (LVDP) and coronary perfusion pressure (CPP), a measure of coronary resistance, were measured in isolated saline perfused hearts. As shown in Figure 4a, CPP was not different between TG and WT hearts under basal conditions. However, infusion of l-arginine (200 μmol/L) caused CPP to decrease in TG but was without effect in WT hearts. The changes in coronary resistance elicited by l-arginine were due to stimulation of NOS since d-arginine (200 μmol/L) did not influence CPP. Furthermore, ETU, a potent NOS inhibitor, fully blunted the l-arginine–induced decrease of CPP in TG hearts (data not shown). LVDP tended to be lower in TG hearts under all conditions tested, and this difference became significant when NO release was elevated by l-arginine infusion (Figure 4b).
To assess the mitochondrial respiratory chain function, NMR analysis of high-energy phosphates was performed. However, we found no significant differences between WT and TG mice regarding phosphocreatine content (Figure 4c) and oxygen consumption (Figure 4d). Similarly, no differences in ATP, pHi, and Pi were observed (data not shown).
Measurement of iNOS Activity In Vivo
To estimate the activity of iNOS in intact hearts, coronary effluent perfusate of isolated iNOS-overexpressing hearts was collected to measure release of the two major NO oxidation products, nitrate and nitrite. Using standard perfusion buffer, NO3− release by TG36 hearts was below detection limit, but was substantially elevated above background to 20.4 nmol · min−1 · g−1 wet weight (w.w.) in the presence of in vivo–like l-arginine concentrations (200 μmol/L, n=6). This effect was fully abolished by ETU (100 μmol/L). In contrast, NO3− release by WT hearts did not increase above background levels under all conditions tested.
Coronary venous release of basal NO2− in WT and TG hearts was 21.8±3.2 and 33.9±9.4 pmol · min−1 · g−1 w.w., respectively (P<0.05, n=3). In the presence of l-arginine, NO2− significantly increased in TG hearts to 61.0±5.8 pmol · min−1 · g−1 w.w. (P<0.0001, n=3), while remaining largely unchanged in WT hearts (26.4±5.9 pmol · min−1 · g−1 w.w. [n=3]). The finding that NO2− release is 340 times lower than that of NO3− suggests that NO in the heart was mainly converted to nitrate. When relating total cardiac iNOS activity in vitro under optimized conditions (citrulline assay: 550 pmol · min−1 · mg−1 protein) to the l-arginine–induced release of the NO oxidation products (nitrate+nitrite: 70 pmol · min−1 · mg−1 protein) into the coronary venous effluent in vivo, it can be calculated that the actual iNOS activity amounted to ≈12% of the enzymes measured under Vmax conditions. Although this fraction appears to be low, 12% from total in vitro activity amounts to 70 pmol · min−1 · mg−1 protein, it is still 40 times above WT controls (1.7 pmol · min−1 · mg−1 protein).
Consistent with these ex vivo measurements, cardiac NOx levels were significantly elevated in iNOS transgenic mice (WT: 5.8±4.2 nmol · g−1 w.w.; TGiNOS: 14.8±5.2 nmol · g−1 w.w.; n=6, P=0.025). In addition, we found a significant increase of NOx levels in the blood of TGiNOS mice (WT: 13.7±3.8 nmol/mL; TGiNOS: 28.9±7.8 nmol/mL; n=6, P=0.013).
NOS Activity and Substrate Supply
To identify mechanisms involved in the attenuation of iNOS activity, the role of cofactors was analyzed. NADPH levels were not different between WT and TG hearts (179±23 [n=3] versus 157±57 [n=3] nmol · g−1 w.w., respectively). Furthermore, when the citrulline assay was performed with and without calmodulin supplementation, no significant differences were found (+calmodulin: 853±106 [n=6] pmol · min−1 · mg−1 protein; −calmodulin: 758±108 [n=6] pmol · min−1 · mg−1 protein).
The finding that supplementation of the perfusion medium with l-arginine caused NO-dependent coronary dilation in the TG hearts only suggested that a limited substrate availability might be of functional importance. Amino acid analysis indeed revealed major differences in the tissue levels of l-arginine and l-citrulline. As evident from Figure 5a, l-arginine levels in TG hearts were only 15% of that in the WT; l-citrulline, on the other hand, was 20-fold above respective controls. This effect was specific for the heart since l-arginine and l-citrulline levels were not different between WT and TG mice in skeletal muscle (l-arginine: 162 versus 160 nmol · g−1 w.w.; l-citrulline: 149 versus 158 nmol · g−1 w.w.; n=4), liver (l-arginine: 7.3 versus 8.4 nmol · g−1 w.w.; l-citrulline: 16.7 versus 19.2 nmol · g−1 w.w.; n=4), and kidney (l-arginine: 86 versus 91 nmol · g−1 w.w.; l-citrulline: 23 versus 22 nmol · g−1 w.w.; n=4). Analysis of serum samples for l-arginine and l-citrulline likewise did not reveal any differences between WT and TG (Figure 5b). All other amino acids were in the normal range both in WT and TG (see the Table in the online data supplement available at http://www.circresaha.org).
To examine whether reduced l-arginine levels in TG hearts might be caused by reduced l-arginine uptake, expression of the cationic amino acid transporters CAT1 and CAT2 were analyzed. RT-PCR analysis (Table 2) revealed that CAT1 expression was generally higher than CAT2, but neither CAT1 nor CAT2 expression was decreased in TG hearts.
In additional experimental series, we analyzed the amino acid content in TG hearts after coronary infusion of l-arginine (200 μmol/L) and ETU (100 μmol/L) for 30 minutes. Interestingly, NOS inhibition corrected l-arginine levels back to control values (200±50.5 nmol · g−1 w.w. [n=3]).
Because the ratio of l-arginine to l-citrulline tissue levels changed from 1:1 in WT to 1:100 in TG, we determined whether the observed l-arginine/l-citrulline ratio may have influenced iNOS activity. In the citrulline assay, a 100-fold increased concentration of l-citrulline significantly decreased iNOS activity by ≈30% from 350±127 (n=6) to 251±129 (n=6) pmol · min−1 · mg−1 protein in vitro.
We further addressed the question whether the observed changes in l-arginine/l-citrulline levels might have been the result of a decreased expression or activity of enzymes known to recycle l-citrulline to l-arginine. RT-PCR for argininosuccinate synthetase (ASS) (Table 2) revealed no difference between WT and TG hearts. Similarly, ASS activity measured in cardiac protein extracts was unaltered (WT=0.145±0.021 nmol argininosuccinate · min−1 · mg−1 protein, n=6; TG=0.148±0.046 nmol argininosuccinate · min−1 · mg−1 protein, n=5).
This study clearly demonstrates that a massive cardiospecific overexpression of iNOS in transgenic mice is not associated with deleterious effects on cardiac hemodynamics and energetics and does not result in heart failure. Obviously, transgenic mice subjected to a normal life cycle tolerated the prolonged overexpression without major functional impairment.
Overexpression of iNOS in cardiac myocytes caused a 2.5-fold increase in cardiac NOx levels. In addition to local overexpression in the heart, we found to our surprise also systemic NOx levels to be elevated 2-fold. These changes most likely reflect the at least 40-fold higher cardiac NOS activity under in vivo–like conditions. Despite this high rate of NO formation, the functional consequences were found to be rather mild. Transgenic animals had no obvious phenotype: they bred normally and showed normal physical activity. Mean arterial pressure was reduced by 8 mm Hg and there was a small decrease in cardiac output. Similarly, we also found a small decrease in left ventricular pressure development in isolated hearts, when perfusion medium was supplemented with l-arginine. Most likely, this is a consequence of enhanced NO production due to high iNOS activity. On the other hand, hearts from TG mice did not differ from WT controls with respect to cardiac oxygen consumption and high-energetic phosphates.
The benign phenotype is surprising in view of the well-known interaction of NO with mitochondrial cytochromes.7 It is important to note that NO released from TG hearts was found to be almost completely converted to nitrate, which is the reaction product of NO with transition metals or oxygenated heme proteins such as oxyhemoglobin or oxymyoglobin.33 Since hearts were perfused with a saline medium in the absence of hemoglobin, myoglobin appears to be a likely candidate for intracellular NO metabolism. In fact, we have recently reported myoglobin to serve as an effective scavenger of NO in the heart resulting in the formation of metmyoglobin and nitrate.27 Thus, the effective degradation of iNOS-derived NO by myoglobin seems to be an important safeguard protecting cardiac mitochondria from NO. Myoglobin should be particularly effective in the present experiments, because both myoglobin and iNOS are localized in the cytosol and therefore the site of NO production and inactivation are almost identical. Lack of sufficient intracellular heme proteins, such as myoglobin, to scavenge NO may explain that iNOS overexpression in pancreatic β cells of transgenic mice caused major functional consequences34 not observed in cardiac muscle.
Comparison of NOS activity in vivo with that measured in vitro revealed that in the heart the overexpressed enzyme was running far below Vmax. This may result from at least three mechanisms: limited availability of substrate,35 product inhibition of the enzyme,36,37⇓ and reduction of cofactors.38 l-arginine is the essential substrate for iNOS, and it has been recognized that the extracellular availability of this amino acid in some35,39⇓ but not in all systems40 can influence cellular rates of NO synthesis. In our experiments, we found no changes in serum levels of l-arginine. Interestingly, however, the intracellular concentration of l-arginine was drastically reduced in iNOS-overexpressing hearts amounting to only 15% of respective controls, whereas l-citrulline was elevated about 20-fold; this shift in amino acid levels was cardiac specific.
It is known from previous studies that a prerequisite for full catalytic activity of iNOS in vivo is the coordinated upregulation of proteins involved in l-arginine supply.39,41⇓ Lipopolysaccharide and cytokines not only stimulate iNOS expression but also are known to cause the concomitant upregulation of l-arginine transporters and/or enzymes of the l-arginine/l-citrulline cycle41,42⇓ involved in resynthesis of l-arginine from l-citrulline via ASS and argininosuccinate lyase (ASL). In our system, however, we found the expression of the cationic amino acid system y+, CAT1 and CAT2, to be unaltered in TG hearts and likewise measurement of the rate-limiting enzyme for arginine recycling, argininosuccinate synthetase, revealed no difference between WT and TG hearts both at the RNA and protein levels. The shift in cardiac amino acid levels is therefore most likely the result of high iNOS activity and not a transporter-dependent process, because pharmacological NOS inhibition in the beating heart rapidly normalized tissue levels of l-arginine.
Aside from limited l-arginine availability, elevated l-citrulline at the concentration measured in transgenic hearts was found to inhibit iNOS activity by ≈30%. Product inhibition by l-citrulline might therefore contribute to the difference between iNOS activity in vivo and in vitro. In addition, there is evidence for feedback autoregulation by NO when the NOS is treated with NO donors.36,37⇓ Furthermore, l-citrulline has been reported to inhibit l-arginine transport in activated macrophages.43 The reduction of in vivo iNOS activity in TG hearts may therefore be the result of at least two factors: a limited pool size of intracellular l-arginine, which cannot be replenished at the rate NO is formed, and feedback inhibition by l-citrulline and NO.
As to the availability of cofactors for iNOS, we found no evidence for a lack of calmodulin. We also found no changes in tissue levels of NADPH, which provides electrons for the formation of NO. Thus, lack of cofactors is unlikely to explain the reduced iNOS activity in vivo.
The data presented here have important implications for the validity of the hypothesis that myocardial induction of iNOS may represent a crucial event in the pathomechanism leading to heart failure. Several clinical and experimental studies revealed that elevated iNOS levels are frequently associated with DCM,12 myocarditis,16 ischemic heart disease, and valvular heart disease.15 Overexpression of iNOS was also found in human cardiac allografts,18 and the expression levels were found to correlate with ventricular contractile dysfunction. However, most of these studies measured only iNOS expression by RT-PCR or immunological techniques; no study so far measured the actual in vivo activity of iNOS in the failing heart. iNOS measured in human myocardial biopsies from patients with DCM4,14⇓ in vitro revealed activities that were ≈20- to 80-fold lower than respective values found in the present study. Since we did not find severe cardiac dysfunction despite NO production being by far higher than that measured in all previous studies, our finding casts serious doubt on the view that iNOS is the crucial factor in the development of heart failure. Expression of iNOS may at best be an index or one element among others in the complex pathophysiological situation of heart failure. As such, our study does not exclude the possibility that in the transition from cardiac hypertrophy to heart failure induced, for example, by biomechanical stress, iNOS-overexpressing mice may develop failure at an earlier stage compared with WT controls. In fact, it was recently shown that iNOS−/− mutants show a significant increase of survival after myocardial infarction,44,45⇓ suggesting a role of NO at a late stage of this disease.45 On the other hand, cytokines can lead to cardiac dysfunction without iNOS induction,46 which is in support of the view that iNOS expression may only be one factor of a complex set of pathomechanisms leading to heart failure.
This work was supported by Deutsche Forschungsgemeinschaft SFB 242–Teilprojekt A5 and Volkswagenstiftung Projekt I75470. The authors would like to thank B. Patzer and L. Bohne for excellent technical assistance, S. Küsters for technical help, and P. Niesswand and W. Teutscher for animal care.
Original received April 12, 2001; revision received November 13, 2001; accepted November 14, 2001.
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