Oxidized Low-Density Lipoprotein–Dependent Endothelial Arginase II Activation Contributes to Impaired Nitric Oxide Signaling
Oxidized low-density lipoprotein (OxLDL) impairs NO signaling and endothelial function, and contributes to the pathogenesis of atherosclerosis. Arginase reciprocally regulates NO levels in endothelial cells by competing with NO synthase for the substrate l-arginine. In human aortic endothelial cells, OxLDL stimulation increased arginase enzyme activity in a time- and dose-dependent manner. Arginase activity reached its maximum as early as 5 minutes, was maintained for a period of more than 48 hours, and was associated with a reciprocal decrease in NO metabolite (NOx [nitrite and nitrate]) production. Furthermore, OxLDL induced arginase II mRNA expression after 4 hours. Small interfering RNA targeted to arginase II decreased both the quantity and the activity of arginase from baseline, prevented OxLDL-dependent increases in arginase activity, and induced an increase in NOx production. Immunofluorescence analysis revealed an association of arginase II with the microtubule cytoskeleton. Microtubule disruption with nocodazole caused a dramatic redistribution of arginase II to a diffuse cytosolic pattern, increased arginase activity, and decreased NOx production, which was restored in the presence of the specific arginase inhibitor (S)-(2-boronoethyl)-l-cysteine (BEC). On the other hand, epothilone B prevented microtubule disruption and inhibited OxLDL-dependent increases in arginase activity and attenuated OxLDL-dependent decreases in NOx. Preincubation of rat aortic rings with OxLDL resulted in an increase in arginase activity and a decrease in NOx production. This was reversed by arginase inhibition with the BEC. Thus, OxLDLs increase arginase activity by a sequence of regulatory events that involve early activation through decreased association with microtubules and a later increase in transcription. Furthermore, increased arginase activity contributes to OxLDL-dependent impairment of NOx production. Arginase, therefore, represents a novel target for therapy in atherosclerosis.
The endothelium plays a central role in overall vascular homeostasis by regulating vasoreactivity, platelet activation, leukocyte adhesion, and smooth muscle cell proliferation and migration. Endothelial nitric oxide (NO), an important vasoprotective molecule, is a major modulator of these effects, and impaired NO signaling with associated endothelial function is considered an early marker of the atherogenic process.
Nitric oxide is produced by the action of endothelial NO synthase (eNOS), which uses l-arginine as its substrate. However, l-arginine is also a substrate for arginase, which converts l-arginine to l-ornithine and urea. Thus, substrate availability to, and thereby enzymatic products of, either enzyme may be influenced by the activity and substrate utilization of the other. Specifically, increased arginase activity and l-arginine utilization may limit l-arginine bioavailability to NOS and thus compromise NO production1,2 and increase O2 production by NOS uncoupling.3
Arginase is present in 2 isoforms: arginase I, or the hepatic isoform; and arginase II, or the extrahepatic (mitochondrial) isoform; each of which is encoded by a distinct gene.4,5 Arginase I catalyzes the final step of the urea cycle in hepatocytes. However, recent studies in other tissues demonstrate that arginase I expression can be induced by lipopolysaccharide (LPS), interleukin (IL)-13, and hypoxia.6–10 l-Ornithine, the product of arginase II, is essential in the synthesis of polyamines that modulate cell proliferation and differentiation.11 Importantly, both arginase isoforms have been shown to reciprocally regulate NO production. Arginase I regulates NO production in rat aortic endothelial cells12 and macrophages.13 Arginase II reciprocally regulates penile NO production, modulating erectile function,14 and is upregulated by thrombin stimulation in human umbilical vein endothelial cells (HUVECs) via a Rho pathway–dependent mechanism.15
These studies indicate that arginase has the capacity to reciprocally regulate NO production. However, the specific pattern of arginase isoform/s expression in human aortic endothelial cells (HAECs) and its/their mechanism of activation are completely unknown. OxLDLs are pivotal molecules in initiating and propagating the atherosclerotic process, an early seminal feature of which is decreased NO bioavailability. However, the role of arginase in limiting NO in HAECs, the specific arginase isoform involved and the potential effect of OxLDLs on arginase expression, have not been previously investigated. Thus, we tested the hypothesis that OxLDL causes upregulation of arginase in HAECs, leading to impaired endothelial NO production, an important pathophysiological mechanism underlying atherogenesis.
Materials and Methods
HAECs were maintained according to the protocol of the supplier (Intracel Co, Frederick, Md) and incubated with starvation medium for 24 hours before OxLDL treatment.
Arginase Activity Assay
Arginase activity was determined as described previously.12
Transfection of Arginase II Small Interfering RNA
Cells were transfected using oligofectamine reagent, incubated 36 hours in the growth medium, and then serum starved before stimulation with OxLDL.
Western Blot Analysis
Cell extracts were subjected to SDS-PAGE and analyzed the densitometry of bands with NIH ImageJ.16
Total tubulin was immunoprecipitated using polyclonal antisera, with modifications of previously described techniques.17
Total RNA was prepared using TRIzol Reagent. PCR was performed in an iCycler optical system using SYBR green PCR master mix.
The treated cells were fixed and incubated with specific antibody, and images were acquired using a epifluorescence microscope.
Tubulin Depolymerization Assay
Tubulin depolymerization was evaluated by a simple method, as described previously by Giannakakou et al.18
All data are reported as mean±SEM. Statistical significance was determined by 1-way ANOVA.
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
OxLDL Stimulation Increases Arginase Activity in HAECs
We first wished to determine the effects of OxLDL on arginase activity in cultured HAECs. OxLDL (50 μg/mL) stimulation induced a time-dependent increase in arginase enzyme activity (Figure 1A). Arginase activity was increased as early as 5 minutes (1.6-fold increase versus control, P<0.001) and persisted for 48 hours. The maximal arginase activity was observed 10 minutes after OxLDL stimulation (2.0-fold increase versus control, P<0.001). In addition, the dose response of this OxLDL effect was measured in HAECs at 10 minutes. The enzyme activity gradually increased in a dose-dependent manner (Figure 1B), with a maximal effect at 100 μg/mL of OxLDL (1.6-fold versus control, P<0.001). Because the submaximal responses occurred at 50 μg/mL (OxLDL 50 μg/mL versus 100 μg/mL, P<0.045), this concentration was used for all subsequent experiments. The dose of 25 to 50 μg/mL is within the order of magnitude one would observe clinically.19 NOx metabolite (NOx [nitrite and nitrate])
OxLDL Stimulation Reciprocally Decreases NO Production
Given recent data suggesting that arginase reciprocally regulates NOS activity by limiting l-arginine bioavailability,1 we tested whether an OxLDL-induced increase in arginase activity was associated with a decrease in NO metabolite (NOx [nitrate and nitrate]) measurement. Furthermore, because decreased eNOS expression has been shown in endothelial cells exposed to OxLDL, we also assayed total eNOS following OxLDL stimulation. As demonstrated in Figure 2, OxLDL stimulation resulted in a time-dependent decrease in NOx production starting as early as 4 hours. This decrease continued to a minimum level of 34% of baseline (OxLDL versus control, P=0.002) at 48 hours. The total amount of eNOS protein remained constant until between 12 and 24 hours. Interestingly, arginase inhibition with BEC (10 μmol/L) prevented the OxLDL-dependent decrease in NOx production at all time points starting as early as 4 hours (102%, OxLDL+BEC versus control=14.6±0.9 versus 14.2±1.0). Thus, the OxLDL-dependent decrease in NOx production occurred before the decline in the abundance of eNOS. Furthermore, arginase inhibition prevented the OxLDL-dependent decrease in NOx production at all time points despite the decrease in eNOS levels seen at 12 hours. BEC treatment alone had no effect on baseline HAEC NOx production.
Arginase II Is the Key Isoform Regulating Arginase Activity and NOx Production in HAECs
Because both isoforms of arginase have been demonstrated to regulate NOx production, we wished to determine which isoform was responsible for this effect in HAECs. RT-PCR was performed with specific primer sets for arginase I and II. As demonstrated in Figure 3A, the expression of arginase II was identified by its RT-PCR product in both control and OxLDL-stimulated HAECs. On the other hand, arginase I expression was not detected in either stimulated or nonstimulated HAECs, indicating that arginase I was not induced by OxLDL at all time points. Given that only arginase II was expressed in HAECs, we wished to confirm the functional role of this isoform. Because of the lack of isoform-specific arginase inhibitors, we used a small interference RNA (siRNA) technique to perform loss-of-function experiments. siRNA targeted to arginase II was transfected into HAECs by Oligofectamine reagent. siRNA expression significantly decreased arginase II protein expression (Figure 3B) to 72% (100±5.3 versus 73.0±3.3: control versus 25 μmol/L siRNA; P<0.005) of baseline. Decreased protein levels were associated with a proportional decrease in arginase enzyme activity. As demonstrated in Figure 3C, siRNA at the concentration of 6.6 pmol/L decreased enzyme activity from 193.2±26.7 (OxLDL stimulated group) to 93.2 pmol of urea per mg protein per minute. The arginase activity was further decreased (57.8±7.7 pmol urea/mg protein per minute) by increasing the concentration of arginase II-targeted siRNA to 25 pmol/L (P<0.005). Furthermore, NO production was increased from 10.6±0.1 to 13.7±1.2 at 25 pmol/L siRNA arginase II. This represents a 1.6-fold increase compared with the untreated control (8.7±0.8 versus 13.7±1.2, P<0.005). Thus, arginase II appears to be the predominant isoform responsible for reciprocal regulation of NOx production in HAECs. Furthermore, knockdown of arginase II can prevent OxLDL-induced increases in HAEC arginase activity.
Transcriptional Induction and Translational Activation of Arginase II by OxLDL
Given the time-dependent increase in arginase II activity following OxLDL stimulation, we wished to determine the molecular mechanism underlying this phenomenon. We first determined whether OxLDL increased the available pool of arginase II at a transcriptional level. Quantitative PCR was performed at different time intervals. As seen in Figure 4, there was a significant increase in arginase II mRNA transcription at 4 hours (≈2.5-fold, P<0.001). This was completely blocked by the transcriptional inhibitor actinomycin D (10 μg/mL). The induced levels of arginase II mRNA were also translated to protein after OxLDL stimulation for 4 hours and significantly increased after 12-hour stimulation (1.8-fold, P<0.005). This was completely blocked by coincubation of cells with the translational inhibitor cycloheximide (10 μmol/L).
It is important to note, however, that increases in arginase activity as early as 5 minutes following OxLDL stimulation cannot be accounted for by alterations in transcription or translation. This suggests a posttranslational mechanism for the early activation of arginase II following OxLDL stimulation of HAECs.
Dissociation of Arginase II From Microtubules Is a Key Mechanism of Arginase Activation
The rapid activation of arginase II following OxLDL stimulation indicates that changes in the availability or activation state of the enzyme may be occurring. We reasoned that regulation of arginase activity may involve changes in cytoskeletal association. Therefore, we used immunofluorescence imaging to map the topography of arginase II with respect to both actin microfilaments and microtubules in HAECs. Imaging revealed no correspondence between arginase II and the actin cytoskeleton, but a striking colocalization with microtubules (stained with an anti–β-tubulin antibody) was seen (Figure 5). This association of arginase II with microtubules was partially disrupted by OxLDL. To further investigate the dependence of arginase II distribution on the structure of microtubular networks, we depolymerized microtubules with nocodazole (50 μmol/L). Microtubule depolymerization caused a dramatic redistribution of arginase II to a diffuse cytosolic pattern. Thus, the dissociation of arginase from the microtubules may represent a novel molecular activation mechanism.
To quantitate the dependence of OxLDL-mediated increases in arginase activity on release from microtubule association, we performed tubulin depolymerization assays (see Materials and Methods). Briefly, cell lysates were separated into an insoluble, polymerized tubulin fraction and a soluble, depolymerized tubulin fraction. The total amounts of tubulin, arginase II, and arginase activity were measured in each fraction. OxLDL treatment led to a significant redistribution of tubulin from the insoluble fraction to the soluble fraction within 5 minutes after treatment (Figure 6A). This tubulin redistribution was also accompanied by a concomitant redistribution of arginase II to the soluble fraction and an increase in arginase activity. Similar results were seen using nocodazole as a positive control. Thus, arginase II was found primarily in association with the microtubule cytoskeleton fraction in untreated cells, where its activity appeared to be constrained, but it was redistributed to the soluble (cytosolic) fraction and activated on OxLDL stimulation. Thus, OxLDL appeared to increase arginase activity by inducing microtubule depolymerization and release of the enzyme into the cytosol. To further confirm whether an interaction of arginase II and tubulin exists, cell lysates solubilized in RIPA buffer after tubulin stabilization were used for immunoprecipitation with anti–tubulin-specific antibody (Figure 6B). As predicted, arginase II was coimmunoprecipitated with tubulin in immunoblot analysis, but not in negative control without tubulin antibody.
Indeed, nocodazole treatment increased arginase activity in a dose-dependent manner (40.4±7.6, 100.7±11.2, and 134.3±0.3 pmol urea/mg protein per minute), respectively, in control conditions and at 5 μmol/L and 50 μmol/L nocodazole, P<0.001). Nocodazole in combination with OxLDL increased arginase to a level (141.9±7.1) that was statistically different from OxLDL alone, suggesting that they may act by a partially different mechanism (Figure 6C, top). Nocodazole treatment also led to a reciprocal decrease in NOx (Figure 6C, bottom). Arginase inhibition in the presence of nocodazole restores endothelial cell NO toward that of untreated cells. This is consistent with the notion that arginase activation contributes to a decrease in endothelial cell NO. We next used epothilone B to stabilize the microtubules by halting depolymerization. Epothilone B (0.1 μmol/L) markedly inhibited OxLDL-induced arginase activation (Figure 6D, top). Thus, OxLDL alone showed arginase activity of 125.7±6.9 (pmol urea/mg protein per minute), and HAECs that were treated with both OxLDL and epothilone B showed arginase activity of 73.6±2.8 (pmol urea/mg protein per minute) (OxLDL+epothilone B versus OxLDL, P<0.005). Epothilone B alone resulted in a small increase in basal endothelial cell NOx that was not statistically significant. Epothilone B, although completely blocking OxLDL dependent arginase activity, attenuated but did not completely block OxLDL-mediated decreases in endothelial NOx production (6.8±0.1 [OxLDL] versus 8.1±0.5 [OxLDL+ epothilone B]; P<0.05). This suggests that prevention of dissociation of arginase by OxLDL is unlikely to be the sole mechanism by which epothilone B may modulate endothelial NO signaling. Thus, both OxLDL treatment and nocodazole-induced microtubule depolymerization, resulted in increased arginase II activity and decreased NOx production, which was restored with arginase inhibition. Epothilone B–dependent stabilization of the microtubular structure prevented OxLDL-dependent activation of arginase II and attenuated the decrease in NOx production. This lends further support to our hypothesis that OxLDL-dependent arginase II activation is mediated by arginase II dissociation from microtubules and that this, in part, contributes to decreased endothelial cell NO production.
Arginase Activation and Reciprocal NOx Decrease in OxLDL-Treated Rat Aorta
We next tested whether OxLDL activates arginase in an in vitro vascular bioassay. Preincubation of rat aortic rings with OxLDL (16 hours) resulted in a significant increase in arginase activity in aortic rings in which the endothelium remained intact (E+) (n=5) but not in rings in which the endothelium had been denuded (E−) (n=4) (Figure 7A). This is consistent with our previous observations confirming that arginase is confined primarily to the endothelium in vascular tissue.12 Furthermore, increased arginase activity was associated with a reciprocal decrease in NOx production in E+ rings. (Figure 7C). On the other hand, preincubation of the E+ rings with the arginase-specific inhibitor BEC decreased arginase activity (Figure 7B) and increased vascular NO production following OxLDL treatment (Figure 7D).
The idea that arginase modulates NOx production by NOS through limiting l-arginine substrate is emerging as a general mechanism for NOS regulation and appears to contribute to the pathobiology of a number of disease processes in which NO is dysregulated.1 Here we demonstrate for the first time that OxLDL, the primary cause of atherosclerotic disease, increases the availability and activity of arginase II (at both transcriptional and posttranslational levels), reciprocally decreases NOx production, and contributes to impaired vascular NO signaling. Furthermore, we describe a novel mechanism for the activation of arginase within the endothelial cell: release of the enzyme from microtubule-mediated constraint (Figure 8).
Endothelial Expression of Arginase
It is increasingly recognized that arginase is constitutively expressed in endothelial cells of multiple vascular beds.5,20–22 It has recently been demonstrated that both isoforms are constitutively expressed in HUVECs, where they regulate progression through the cell cycle (inhibition of arginase leads to growth inhibition).11 The predominant isoform in this cell population appears to be arginase I. In contrast, in a porcine coronary artery model, Zhang et al23 have shown that the arginase I isoform is mainly responsible for limiting endothelial-dependent relaxation. Moreover, bovine pulmonary ECs express both arginase I and II, which can be upregulated by cytokines, and arginase inhibition in these cells accentuates NO release.2 We have previously demonstrated constitutive expression of both arginase I and II in rat aortic endothelium, where arginase I is the predominant isoform.12 Our ongoing studies in the rat aorta (using antisense technology) have demonstrated that arginase I is the isoform responsible for reciprocal regulation of NOS in endothelium and that its function and abundance are increased with aging.1
Our quantitative RT-PCR (qRT-PCR) and Western blot data support the notion that arginase II is the predominant (if not the only) isoform expressed in HAECs. This was further supported by the siRNA experiment in which arginase II-specific siRNA modulated arginase activity and reciprocally enhanced NOS activity. siRNA specific for arginase II decreased protein levels in a dose-dependent manner and dramatically inhibited OxLDL-dependent arginase activation.
Posttranslational Activation of Arginase
Immunofluorescence data and tubulin polymerization assays demonstrate that OxLDL induces the dissociation of arginase II from the microtubule cytoskeleton. Nocodazole causes microtubule depolymerization by quadrupling the rate of GTP hydrolysis on the tubulin dimer24 and dramatically disrupts microtubule structure and function (Figure 5G). Nocodazole also induces the activation of arginase. OxLDL induces a less dramatic degree of microtubule destabilization but still causes net depolymerization of microtubules and arginase activation. Additionally epothilone B, a microtubule-stabilizing agent, prevented both OxLDL-dependent microtubule depolymerization and arginase activation. Taken together, these data suggest that OxLDL activates arginase via a novel mechanism involving disengagement from the microtubule cytoskeleton in a manner that does not lead to complete disruption of microtubule infrastructure. The precise mechanism of this activation event is not clear at this point, but it may involve both a loss of an inhibitory effect of the tubulin-bound state as well as release-dependent interactions with other proteins that enhance its activity. The microtubule cytoskeleton may serve as a critical component of signaling pathway regulation through either protein sequestration or site-directed protein delivery.25 Our data suggest that microtubule-mediated sequestration may regulate the activity of arginase II in HAECs.
The data presented here further indicate that microtubule-dependent regulation of arginase affects NO production in HAECs. Thus, coincubation of cells with OxLDL and epothilone B attenuated OxLDL-mediated suppression of NO production. Furthermore, the decrease in NO production following nocodazole treatment was blocked by the arginase inhibitor BEC. Recent reports describe a cellular strategy for posttranslational regulation of inducible NOS (iNOS) in several different cell types that may lend insight into our findings described.26 In this work, the downregulation of iNOS activity is shown to occur via incorporation of the enzyme into aggresomes in a dynein- and dynactin-dependent process that is abrogated by microtubule disruption with nocodazole. Activation of iNOS is accompanied by release from these aggresomes, which are juxtanuclear and are associated with both the microtubule organizing center and mitochondria. Analogous processing of arginase would be consistent with this reported data and the emerging literature regarding the role of the endothelial cytoskeleton in regulation of eNOS.27,28
Reciprocal Regulation of NOS
Our data demonstrate that arginase activation by OxLDL reciprocally downregulates NO production in a time-dependent manner. This decrease in NO is completely blocked by the arginase inhibitor BEC, despite a time-dependent decrease in levels of total eNOS. This suggests that the decrease in basal NO in endothelial cells by OxLDL is predominantly dependent on arginase upregulation. This finding is consistent with the idea that OxLDL may result in eNOS uncoupling; in which case, O2 is produced by the enzyme instead of NO. This results from electrons flowing from the reductase domain in the heme to molecular oxygen rather than l-arginine. There are a number of circumstances in which this may occur, specifically tetrahydrobiopterin cofactor deficiency as well as a relative deficiency states for l-arginine.29 It is our primary thesis that the upregulation of arginase contributes to this mechanism and that the inhibition of arginase therefore restores NO production. Thus, it is the availability of substrate cofactors and local eNOS microdomain concentrations of l-arginine rather than the expression level/abundance of the eNOS enzyme that is critical in NO production. Furthermore, decreased NO production and its restoration with arginase inhibition (BEC) precede the decrease in eNOS expression following OxLDL stimulation. These data support this alternate mechanism, whereby OxLDL impairs nitroso-redox balance in the ECs.
Other proposed mechanisms for OxLDL-induced decrements in NO availability include the upregulation of caveolin-1 expression followed by eNOS sequestration, increased ROS production with subsequent decreased NO bioavailability,30 and decreased eNOS activity via inhibition of protein kinase Cα–mediated phosphorylation of eNOS threonine 495.31 An additional factor in the reciprocal regulation of eNOS activity by arginase may be subcellular compartmentalization. eNOS is known to bind to the scaffolding protein caveolin-1. This is the structural backbone of the plasma membrane invaginations known as caveolae, which have several well-described signal transduction functions.32 Caveolae are shuttled along microtubules from the cell periphery and plasma membrane-proximal sites to perinuclear sites neighboring the microtubule organizing center and nocodazole-induced microtubule depolymerization is associated with a dramatic increase in the membrane-associated pool of caveolin-1.33–35 Dissociation of arginase from microtubule-dependent mechanisms may bring it into proximity of l-arginine pools that are shared by eNOS and were not available to arginase when the latter was bound to tubulin. This phenomenon may indeed explain the time course of decreased NO production. Although arginase activity is increased rapidly, it is only after 4 hours that NO production begins to decrease. This may represent a time-dependent depletion of the NOS-accessible l-arginine pool by arginase to a point at which l-arginine becomes substrate limiting.
Transcriptional Regulation of Arginase
We have clearly demonstrated 2 temporally distinct mechanisms responsible for the activation of arginase. One involves dissociation from the microtubules, and the other involves an increase in mRNA transcription leading to an increase in protein levels. There are few studies that interrogate the factors involved in the transcription of arginase. Arginase I expression has been shown to be upregulated in wound-derived fibroblasts and rat aortic smooth muscle cells following stimulation with transforming growth factor-β and IL-4, IL-4, and IL-13. Furthermore, arginase I expression appears to be regulated by the transcription factors CTF/NF-1, Sp1, and C/EBP.36 With regard to arginase II, LPS stimulation induces its expression in rat aortic endothelial cells and macrophages, but the involved transcriptional factors remain to be elucidated.21 Thus in pathophysiological scenarios such as sepsis, arginase I and arginase II are coinduced with iNOS following LPS administration, leading to speculation that arginase I may limit sustained overproduction of NOS.
Rescue of Vascular Endothelial NO Production
As predicted, incubation of OxLDL with endothelialized rat aortic rings resulted in a significant impairment in endothelial NO production. This is consistent with a plethora of in vivo and in vitro data in humans37 and animals38 demonstrating that impairment of endothelial function and NO production is a hallmark of OxLDL-mediated atherosclerotic disease. Our data demonstrating that inhibition of arginase restores vascular endothelial NO production following OxLDL treatment strongly suggest that arginase may represent a therapeutic target for OxLDL-dependent endothelial dysfunction.
In conclusion, we have demonstrated that OxLDL increases arginase II activity by 2 mechanisms: (1) a novel early posttranslational event involving the release of the enzyme from microtubule association and resulting in activation; and (2) transcriptional upregulation. Arginase II upregulation reciprocally decreases endothelial NO production and NOS activity. Finally, these changes in NO processing can be restored by either genetic or pharmacological inhibition of arginase II. Thus, arginase represents a therapeutic target for OxLDL-dependent endothelial dysfunction, the hallmark of atherosclerotic vascular disease.
Sources of Funding
This work was supported by NIH grants AG021523 (to D.B.), HL058064 (to L.R.), and AI061042 (to L.R.); a grant from the National Space Biomedical Research Institute through the National Aeronautics and Space Administration CA00405 (to A.S. and D.B.); and the Johns Hopkins Funds for Medical Discovery (to L.R.).
This manuscript was sent to Donald D. Heistad, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
↵*Both authors contributed equally to this work.
Original received February 23, 2006; resubmission received June 15, 2006; revised resubmission received August 10, 2006; accepted September 14, 2006.
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