Peroxynitrite Causes Endoplasmic Reticulum Stress and Apoptosis in Human Vascular Endothelium
Implications in Atherogenesis
Objective— Peroxynitrite, a potent oxidant generated by the reaction of NO with superoxide, has been implicated in the promotion of atherosclerosis. We designed this study to determine whether peroxynitrite induces its proatherogenic effects through induction of endoplasmic reticulum (ER) stress.
Methods and Results— Human vascular endothelial cells treated with Sin-1, a peroxynitrite generator, induced the expression of the ER chaperones GRP78 and GRP94 and increased eIF2α phosphorylation. These effects were inhibited by the peroxynitrite scavenger uric acid. Sin-1 caused the depletion of ER–Ca2+, an effect known to induce ER stress, resulting in the elevation of cytosolic Ca2+ and programmed cell death (PCD). Sin-1 treatment was also found, via 3-nitrotyrosine and GRP78 colocalization, to act directly on the ER. Adenoviral-mediated overexpression of GRP78 in endothelial cells prevented Sin-1–induced PCD. Consistent with these in vitro findings, 3-nitrotyrosine was observed and colocalized with GRP78 in endothelial cells of early atherosclerotic lesions from apolipoprotein E–deficient mice.
Conclusions— Peroxynitrite is an ER stress-inducing agent. Its effects include the depletion of ER–Ca2+, a known mechanism of ER stress induction. The observation that 3-nitrotyrosine–containing proteins colocalize with markers of ER stress within early atherosclerotic lesions suggests that peroxynitrite contributes to atherogenesis through a mechanism involving ER stress.
Endothelial dysfunction/injury represents a key early step in atherogenesis. The majority of risk factors for atherosclerosis, including, hyperlipidemia, hypertension, diabetes, and smoking, are associated with endothelial dysfunction.1 A major function of the vascular endothelium is the regulation of vascular tone by NO through NO-induced smooth muscle relaxation.2 Reduced NO bioavailability is a common feature in atherosclerosis and can result from oxidative stress.3 In the apolipoprotein E–deficient (apoE−/−) mouse model of atherosclerosis, endothelial dysfunction, as shown by decreased NO-mediated vasodilation to acetylcholine, correlates with increased atherosclerotic lesion size.4 The availability of NO and superoxide (.O2−) within the atherosclerotic lesion creates the conditions for peroxynitrite formation because NO and .O2− react at physiological pH to form peroxynitrite.5 A marker of peroxynitrite generation, 3-nitrotyrosine (3-NT) is elevated in human atherosclerotic lesions.6,7 However, the mechanism by which reduced NO bioavailability and peroxynitrite formation contribute to atherosclerosis remains uncertain.
Endoplasmic reticulum (ER) stress, a cellular stress pathway induced by the accumulation of unfolded proteins in the ER, may offer an explanation for the contribution of peroxynitrite to atherosclerosis. ER stress results in an evolutionary conserved cellular response involving the upregulation of a set of genes, including ER-resident chaperones referred to as the unfolded protein response (UPR). An additional component of the ER stress response is decreased protein biosynthesis mediated by eIF2α phosphorylation. Although the UPR is considered a survival pathway, prolonged or severe ER stress can lead to programmed cell death (PCD) via specific pathways originating from the ER.8 ER stress is associated with atherosclerotic disease,9,10 and substances such as homocysteine (Hcy) that reduce NO bioavailability induce ER stress in endothelial cells11 and have proatherogenic effects.12–14 Atherosclerotic lesions from apoE−/− mice with normal plasma Hcy levels are also immunopositive for ER stress markers, including GRP78/94 and phospho-PERK,10 an ER membrane-bound protein kinase that phosphorylates eIF2α. Given our recent findings that UPR activation occurs at all stages of atherogenesis,15 peroxynitrite may be an important activator of this cellular stress pathway.
In this study, we demonstrate that peroxynitrite induces ER stress in the vascular endothelium, a cell type relevant to atherogenesis. 3-NT staining in endothelial cells suggests that peroxynitrite acts directly on the ER because it colocalized with the ER marker GRP78. Peroxynitrite-induced ER stress results in endothelial PCD, a process that is prevented by overexpression of GRP78. The observation that 3-NT and GRP78 staining are increased and colocalize in early atherosclerotic lesions from apoE−/− mice suggests a role for this mechanism in atherogenesis.
For a full description of the Materials and Methods, please see the online supplement, available at http://atvb.ahajournals.org.
Primary human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were from Clonetics and grown in endothelial growth medium-2 (EGM-2) supplemented with Clonetics SingleQuots.16
HAECs were induced to overexpress human GRP78 via infection with the recombinant adenoviral vector containing human GRP78 cDNA (AdV-GRP78).17 β-galactosidase (AdV-β-gal) infection was used as a viral control.
Animal Model of Atherogenesis
C57BL/6J control (n=4) and apoE−/− female mice (n=4), back-crossed 9 to 10 generations onto a C57BL/6J background, were obtained from Jackson Laboratory at 3 to 5 weeks of age. All mice were fed normal mouse chow (Harlan Teklad) ad libitum with free access to drinking water. All procedures were approved by the McMaster University animal research ethics board.
NO, Superoxide, and Peroxynitrite Measurement
NO was measured using DAF-FM as described previously.18,19 Superoxide measurements were made similarly with the .O2− reporter dye dihydroethidium (DHE). Peroxynitrite was detected through its ability to nitrate protein tyrosine residuals.
Western Blot Analysis
HUVECs or HAECs were cultured for 2, 4, 8, or 18 hour in EGM-2 medium or in medium containing DETA-NoNoate (1 mmol/L), Sin-1 (1 mmol/L), or authentic peroxynitrite (5 to 1000 μmol/L) with or without uric acid (1 mmol/L). Total protein lysates were dissolved in SDS-PAGE sample buffer and separated on 10% SDS-PAGE gels under reducing conditions and transferred to nitrocellulose membranes. Immunoblots were incubated with primary antibodies to GRP78/GRP94 (anti-KDEL), TDAG51, phospho-eIF2α, or 3-NT, followed by the appropriate horseradish peroxidase–conjugated secondary. Membranes were developed with Renaissance Western blot Chemiluminescence reagent.16
Cytosolic and ER–Ca2+ measurements were made at 37°C with fura-2 AM (5 μmol/L) ratiometrically (340 nm/380 nm excitation; 510 emission) on a SpectraMax Gemini Spectrofluorometer (Molecular Devices). Fura-2 responses were calibrated intracellularly as described previously.18,19 The area under the curve (AUC) of the thapsigargin response gave a measure of ER–Ca2+ content.
Cell Death and Apoptosis Assay
Confluent cell cultures were treated for 2, 4, 8, or 24 hours in serum-free EGM-2 medium with either DETA-NoNoate (1 mmol/L) or Sin-1 (1 mmol/L) with or without uric acid (1 mmol/L). The medium was collected for lactate dehydrogenase (LDH) assay as per manufacturer instructions. For TUNEL, cells were fixed in buffered 4% paraformaldehyde and then permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate buffer. In negative controls (minimum), the TdT enzyme was omitted, and in positive controls (maximum), 60 Kunitz/mL DNase I was used to fragment DNA. The TUNEL assay was replicated in 96-well plates for quantification of percent apoptosis in reference to minimum and maximum TUNEL fluorescence.
3-NT and GRP78 Colocalization
Perfusion-fixed specimens from 10- to 12-week-old C57BL/6J and apoE−/− mice were embedded and serial sectioned (4-μm thick) at the level of the aortic root. HAECs grown on cover slips and treated for 4 hours with Sin-1 (1 mmol/L) or aortic root sections were processed for confocal microscopy to determine colocalization of 3-NT with GRP78. Sections and cover slips were incubated with the 3-NT antibody (1:100) and a primary goat anti-GRP78 antibody (1:50). Secondary donkey anti-rabbit Alexa 488 (1:200) and secondary donkey anti-goat Alexa 594 (1:200) were used for 3-NT and GRP78 detection, respectively. Nonspecific background staining was assessed by omitting the primary antibody. Images were captured using a Zeiss LSM 510 confocal microscope.
Values are expressed as mean±SE. Comparison between the means was performed by Student’s unpaired t test. ANOVA was used for multiple comparisons among the means. Significance was recognized at the 95% confidence level.
NO, Superoxide, and Peroxynitrite Generation in Cultured Endothelial Cells
Treatment of HUVECs or HAECs with DETA-NoNoate showed a dose-dependent (10 μmol/L to 1 mmol/L) and time-dependent (30 minutes to 1 hour) increase in DAF fluorescence signal, an indication of NO production (data not shown). DETA-NoNoate treatment (1 mmol/L) for 1 hour significantly increased DAF fluorescence 15.7±0.5-fold (P<0.001) over vehicle-treated cells. Sin-1 (1 mmol/L) treatment for 1 hour also significantly increased DAF signal 9.6±0.3-fold (P<0.001) over vehicle; however, it was significantly less than DETA-NoNoate (Figure 1A; P<0.001). The DAF signal generated by DETA-NoNoate or Sin-1 was inhibited with carboxy-PTIO (1 mmol/L; Figure 1B and 1C; P<0.001), indicating NO was responsible for the increase in DAF signal.
HUVECs or HAECs loaded with the .O2− reporter dye DHE showed a significant 6.0±0.2-fold intensity increase with potassium superoxide compared with vehicle (Figure 1D; P<0.001). DETA-NoNoate (1 mmol/L) treatment for 1 hour did not significantly increase DHE signal, whereas Sin-1 (1 mmol/L) treatment for 1 hour significantly increased DHE signal intensity 2.0±0.2-fold (P<0.01) over vehicle (Figure 1D). The DHE signal generated by 2-hour Sin-1 treatment was inhibited by polyethylene glycol–superoxide dismutase (500 U/mL; Figure 1E and 1F; P<0.001), indicating .O2− was responsible for the increase in DHE signal.
3-NT detection was used to determine whether Sin-1 or DETA-NoNoate produced peroxynitrite in HUVECs. 3-NT labeling showed that 4-hour DETA-NoNoate incubation did not generate peroxynitrite over the 10 μmol/L to 1 mmol/L dose range (Figure 2A). However, 4-hour treatment with Sin-1 significantly increased 3-NT staining 2.6±0.4-fold at 100 μmol/L and 4.6±0.2-fold at 1 mmol/L concentration over vehicle control (ANOVA; P<0.02; Figure 2B). HUVECs incubated with a 1 mmol/L bolus of pure peroxynitrite showed a similar increase (4.2±0.2-fold; P<0.001) in 3-NT staining (Figure 2C). Peroxynitrite-induced generation of 3-NT was significantly inhibited (1.3±0.1-fold; P<0.001) by coincubation with uric acid (Figure 2C). Uric acid was found to exert its inhibitory effects on 3-NT staining by scavenging peroxynitrite because coincubation of uric acid with Sin-1 did not affect the NO signal observed by DAF or the superoxide signal observed by DHE (Figure I, available online at http://atvb.ahajournals.org). Together, these data indicate that Sin-1 treatment generates peroxynitrite in our endothelial cell culture system that can be specifically scavenged by uric acid.
Western blotting was used to assess 3-NT formation in response to peroxynitrite treatment. 3-NT formation was found to be stable over the 2- to 18-hour time period (Figure IIA, available online at http://atvb.ahajournals.org) and occurred within the 50 to 1000 μmol/L peroxynitrite dose range but was absent in the presence of decomposed peroxynitrite (Figure IIB).
Peroxynitrite Induction of ER Stress
Sin-1 (1 mmol/L) treatment in HAECs induced ER stress/UPR activation, as indicated by increased GRP78/GRP94 and TDAG51 protein levels at 18 hours (Figure 3A). Sin-1 treatment also induced the phosphorylation of eIF2α from 4 to 18 hours. These effects were inhibited by coincubation with uric acid (Figure 3A). Thapsigargin (200 nmol/L), a known ER stress inducer that causes ER–Ca2+ depletion, also increased GRP78/GRP94 and TDAG51 protein levels at 18 hours (Figure 3B). Increased eIF2α phosphorylation was observed from 2 to 18 hour. Uric acid did not inhibit thapsigargin-induced ER stress (Figure 3B), indicating that it is not a general inhibitor of ER stress.
Authentic peroxynitrite treatment of HAECs induced eIF2α phosphorylation in a time- and dose-dependent manner that was inhibited by uric acid (Figure IIIA, available online at http://atvb.ahajournals.org). No effect on eIF2α phosphorylation was observed after heat-mediated decomposition of peroxynitrite (Figure IIIB).
Effect of Peroxynitrite on Endothelial Cell Ca2+ Homeostasis
Treatment of HUVECs with DETA-NoNoate (1 mmol/L) for 5 hours did not reduce the thapsigargin response of the ER–Ca2+ pool, as shown by the response AUC (NoNoate 43 900±4700 versus vehicle 42 300±5400 nmol/L per second Ca2+). However, 5-hour treatment with Sin-1 (1 mmol/L) significantly reduced the ER–Ca2+ response (Sin-1 19 200±7500 versus vehicle 42 300±5400 nmol/L per second Ca2+; P<0.05). Positive control treatment with thapsigargin completely abolished ER–Ca2+ response in Ca2+-free medium. Sin-1 (1 mmol/L) treatment in HAECs abolished the thapsigargin response (Sin-1-17 100±13 000 versus vehicle 64100±9700 nmol/L per second Ca2+; P<0.01). However, HAECs exposed to Sin-1 and coincubated with uric acid retained their thapsigargin-mediated ER–Ca2+ response (31 000±5800 nmol/L per second Ca2+).
HAEC cytosolic Ca2+ concentrations [Ca2+]c increased significantly after 5-hour treatment with 1 mmol/L Sin-1 (428±42.5 nmol/L versus 132±111 nmol/L; P<0.05), compared with vehicle. The Sin-1 effect was inhibited by coincubation with uric acid (249±7.8 nmol/L; P<0.05). Similar to Sin-1, treatment with thapsigargin (200 nmol/L) for 5 hours significantly increased [Ca2+]c (430±68.5 nmol/L; P<0.05) over vehicle. For graphic representation of Ca2+ measurements, please see supplemental Figure IV (available online at http://atvb.ahajournals.org).
Effect of Peroxynitrite on Endothelial Cell PCD
To determine whether peroxynitrite induces endothelial cell PCD, LDH release and TUNEL assays were performed. Sin-1 (1 mmol/L) treatment of HAECs significantly increased cell death at 24 hours, compared with control (11±0.5% versus 0±0.5%; P<0.01). DETA-NoNoate had no significant effect, and coincubation with uric acid significantly attenuated Sin-1–induced cell death (1.3±0.7%; P<0.01). TUNEL assay indicated that 24-hour Sin-1 treatment significantly increased PCD in HAECs (33±0.6% versus 9.2±5.5%; P=0.05) over vehicle. This effect was not observed after 24-hour treatment with 1 mmol/L NoNoate or by coincubation of Sin-1 with uric acid (Figure 4A).
Overexpression of GRP78 is cytoprotective after treatment with agents or conditions known to cause ER stress-induced apoptotic cell death.20 To determine whether GRP78 overexpression could protect endothelial cells from peroxynitrite-induced PCD, adenoviral-mediated overexpression of GRP78 in HAECs was performed. AdV-GRP78 increased GRP78 protein levels compared with wild-type (wt) or AdV-β-gal–infected control cells as quantified using a phosphorimager (Figure 4B). GRP78 overexpression (AdV-GRP78; 500 pfu/cell) in HAECs led to a significant decrease in PCD as shown by TUNEL (5.0±2.1% versus 32±3.5% wt or 31±1.6% AdV-β-gal control; P<0.01) in response to 24-hour 1 mmol/L Sin-1 treatment (Figure 4C).
To determine whether Sin-1–induced intracellular peroxynitrite generation acted directly on the ER, HAECs were labeled for GRP78 (Figure 5A) or 3-NT (Figure 5B) after 4-hour Sin-1 (1 mmol/L) treatment. Combined images showed colocalization of 3-NT and GRP78 at the level of the ER (Figure 5C, merged image, arrows). Further experiments were performed to determine whether Sin-1 treatment modified the GRP78 protein through the nitration of tyrosine by immunoprecipitating GRP78 from HAECs and probing for 3-NT on Western blots. In this case, it was determined that Sin-1 (1 mmol/L) treatment of HAECs for 4 hours did not modify GRP78 through the nitration of tyrosine (Figure V, available online at http://atvb.ahajournals.org).
Reactive Nitrogen Species–Induced ER Stress: Role in Atherogenesis
We demonstrated previously that markers of UPR activation are expressed in early atherosclerotic lesions.15 To determine whether reactive nitrogen species (RNS) such as peroxynitrite play a role in atherosclerotic lesion formation, aortic root sections from apoE−/− mice were examined by confocal microscopy. ER stress induction within these early atherosclerotic lesions was examined by detection of GRP78 overexpression, and RNS modification of proteins was detected by 3-NT staining. Confocal microscopy showed that 3-NT (Figure 5D) colocalized with GRP78 (Figure 5E) within the endothelium and macrophage foam cells (arrows) of the early atherosclerotic lesions from apoE−/− mice (Figure 5F, merged image).
The generation of NO and .O2− via Sin-1 treatment established the conditions for peroxynitrite formation in cultured endothelial cells.5 Indeed, we found a dose-dependent increase in 3-NT staining, an established marker of peroxynitrite-mediated protein nitration, in response to Sin-1, but not DETA-NoNoate treatment. These data indicate that DETA-NoNoate treatment increased intracellular NO, whereas Sin-1 treatment increased NO and .O2−, leading to the generation of peroxynitrite.
Consistent with previous studies, Sin-1 treatment induced ER stress/UPR markers as measured by increased GRP78/GRP94 and TDAG51 expression.16,21 Sin-1 treatment also increased the phosphorylation of eIF2α, a downstream marker of PERK activation. Coincubation of Sin-1 with uric acid prevented Sin-1–induced GRP78/GRP94 and TDAG51 expression. We have shown that uric acid blocks ER stress attributable to Sin-1 treatment by scavenging peroxynitrite22 but does not block UPR activation because it had no effect on thapsigargin-induced ER stress.
Sin-1 was shown to deplete thapsigargin-sensitive ER–Ca2+ stores in HUVECs and HAECs. This effect did not occur with DETA-NoNoate treatment and was inhibited by coincubation of Sin-1 with uric acid, indicating that peroxynitrite is responsible for depleting ER–Ca2+ stores in endothelium. It is known that depletion of ER–Ca2+ stores can cause ER stress/UPR activation.21 This appears to be attributable to the Ca2+ dependence of ER protein-folding chaperones including calreticulin and PDI.23 Indeed, the critical role of Ca2+ homeostasis in the ER is supported by previous studies showing that the sarco/ER (SER) Ca2+ ATPase is an ER stress response protein,24 and agents/conditions that disrupt Ca2+ homeostasis induce ER stress and UPR activation.25 We demonstrated this effect by ER stress gene induction in HAECs with the specific SER Ca2+ pump inhibitor thapsigargin (Figure 3B). Thus, abolition of HAEC thapsigargin–stimulated ER–Ca2+ release by Sin-1 is mechanistically sufficient for ER stress induction/UPR activation. The effect of peroxynitrite on Ca2+ uptake by SERCA in porcine coronary artery endothelial cells has been determined and found to produce an IC50 of 302±69 μmol/L.26 These results are consistent with our results in HAECs indicating that pathological concentrations of peroxynitrite are required for complete SERCA inhibition in endothelium. Other effects of peroxynitrite may also contribute to the induction of ER stress in HAECs. These include the ability of peroxynitrite to post-translationally modify proteins and thereby directly affect protein folding and promote protein aggregation27; although peroxynitrite, as detected by 3-NT staining colocalized with GRP78 in the ER, GRP78 was not post-translationally modified by peroxynitrite through tyrosine nitration.
In addition to ER–Ca2+ depletion, Sin-1 treatment resulted in increased resting levels of cytosolic Ca2+. Increased cytosolic Ca2+ appears to be a result of ER–Ca2+ depletion via a process known as capacitative calcium entry.28 The end result of Sin-1 exposure was endothelial PCD, an effect abolished by uric acid. Peroxynitrite-mediated PCD may have resulted from TDAG51 overexpression because TDAG51 has been shown to induce detachment-mediated endothelial PCD.16 The apoptotic process may also be linked to the increase in cytosolic-free Ca2+ because mitochondial Ca2+ overload and cytochrome C release have been shown to be important in PCD,29 in which caspase-3 activation shows Ca2+ dependence30 and stable interactions exist between the ER and a subpopulation of mitochondria, allowing for rapid Ca2+ accumulation.31 In bovine aortic endothelial cells (BAECs), the role of mitochondrial-dependent versus mitochondrial-independent pathways for the induction of PCD by peroxynitrite was examined. The generation of Rho0 BAECs, lacking functional mitochondria, indicated that peroxynitrite induces PCD by caspase-9/mitochondrial-dependent and caspase-8/mitochondrial-independent pathways.32 Peroxynitrite has also been shown to induce DNA damage and activate poly(ADP-ribose) polymerase-I, a DNA nick sensor enzyme. Although peroxynitrite has been shown to directly induce DNA breakage in cell-free systems, Ca2+ chelators appear to prevent this effect in whole cell systems.33
Adenoviral-mediated overexpression of GRP78 in endothelial cells prevented peroxynitrite-induced PCD. This effect may result from the interaction of GRP78 protein with procaspase-7 preventing its activation.34 It may also involve the ability of GRP78 to maintain intracellular Ca2+ homeostasis. GRP78 overexpression provides cytoprotection against hydrogen peroxide–induced increases in cytosolic Ca2+.35 Further, specific inhibition of GRP78 induction by antisense RNA prevented tolerance to intracellular free Ca2+ increase–mediated cell death induced by the oxidant tert-Butylhydroperoxide.36 Together, these data suggest that GRP78 overexpression can prevent cell death attributable to cytosolic Ca2+ overload by buffering Ca2+ in the ER and thereby minimizing Ca2+ fluctuations in the cytosol.
Peroxynitrite, generated by Sin-1 treatment, resulted in all 3 main components of the cellular response to ER stress: (1) upregulation of ER chaperones known as the UPR, (2) the phosphorylation of eIF2α via activation of the PERK pathway, and (3) induction of PCD.8 Consistent with these findings, treatment of microglial cells with Sin-1 also induces the expression of the ER stress proteins CHOP/GADD153 and GRP78/Bip and promotes apoptosis.37 Preconditioning cells by viral-mediated GRP78 overexpression uncoupled peroxynitrite-induced ER stress from PCD, indicating a critical role for this protein in maintenance of cellular homeostasis during pathological RNS generation.
NO also appears to play a role in ER stress gene induction in other cell types. In RAW 264.7 macrophages, lipopolysaccharide/interferon-γ activation and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) incubation caused CHOP/GADD153 expression and apoptosis.38 Also, in pancreatic β-cells, SNAP induces CHOP/GADD153 expression, GRP78 upregulation, and apoptosis.39 These effects of NO on PCD were not observed in HAECs, suggesting a difference in redox potential of the endothelium compared with the other cell types studied in response to NO treatment.
ER stress induction by RNS appears to be important in atherogenesis. Indeed, 3-NT–modified proteins were observed in early atherosclerotic lesions from apoE−/− mice. Further, 3-NT highly colocalized with GRP78 in lesion-resident macrophage foam cells and endothelial cells, suggesting that peroxynitrite or other RNS may be responsible for the observed ER stress in these lesions. Previous findings have indicated that ER stress/UPR activation are prominent features of the atherosclerotic lesion at all stages of development.15 In addition, reduced NO bioavailability3 and RNS stress, as indicated by 3-NT staining,7 have also been found to be hallmark features of atherosclerotic lesions. In this work, we demonstrate a link between RNS stress and ER stress/UPR activation attributable to the effects of peroxynitrite as an ER stress-inducing agent. We also provide novel evidence that overexpression of GRP78 uncouples peroxynitrite-induced ER stress from PCD in endothelium. The identification of pharmacological agents that selectively induce the expression of GRP78 may prove to be a novel therapeutic approach to prevent apoptotic cell death in the developing atherosclerotic lesion, thereby maintaining plaque stability and reducing the possibility of rupture and thrombosis.
This work was supported by research grants to R.C.A. from the Heart and Stroke Foundation of Ontario (T-5385), the Canadian Institutes of Health Research (MOP-74477), and the Ontario Research and Development Challenge Fund. This work was also supported by a Heart and Stroke Foundation of Canada Research Fellowship Award to J.G.D. R.C.A. is a career investigator of the Heart and Stroke Foundation of Ontario.
- Received June 16, 2005.
- Accepted September 15, 2005.
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