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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:2064.)
© 2000 American Heart Association, Inc.

Vascular Biology

Cytokines Induce Upregulation of Vascular P2Y₂ Receptors and Increased Mitogenic Responses to UTP and ATP

Mingyan Hou; Sebastian Möller; Lars Edvinsson; David Erlinge

From the Division of Experimental Vascular Research, Department of Medicine, Lund University Hospital, Lund, Sweden.

Correspondence to Dr David Erlinge, Department of Cardiology, Lund University Hospital, S-221 85 Lund, Sweden. E-mail david.erlinge{at}med.lu.se

	Abstract

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Abstract—P2Y₂ receptors, which mediate contractile and mitogenic effects of extracellular nucleotides in vascular smooth muscle cells (VSMCs), are upregulated in the synthetic phenotype of VSMCs and in the neointima after balloon angioplasty, suggesting a role in the development of atherosclerosis. Because released cytokines in atherosclerotic lesions mediate multiple effects on gene transcription in VSMCs, we speculated that cytokines could be involved in the regulation of P2Y₂ receptor expression. Using a competitive reverse transcription–polymerase chain reaction, we detected that interleukin (IL)-1ß induced a time- and dose-dependent upregulation of P2Y₂ receptor mRNA, which was dramatically enhanced when combined with interferon- or tumor necrosis factor-. Lipopolysaccharide also significantly increased the expression of P2Y₂ receptor mRNA. The upregulation of P2Y₂ receptor mRNA was paralleled at the functional level because IL-1ß significantly increased the UTP-stimulated DNA synthesis and the release of intracellular Ca²⁺. Actinomycin D completely blocked the upregulation of P2Y₂ receptor mRNA expression by IL-1ß, indicating de novo mRNA synthesis. There was no cAMP accumulation in the cells stimulated with IL-1ß. The cyclooxygenase inhibitor indomethacin and the protein kinase C inhibitor RO-31-8220 inhibited IL-1ß–induced upregulation of P2Y₂ receptor mRNA expression, whereas rapamycin and PD098059 had no effects. Furthermore, neither P38 mitogen-activated protein kinase inhibitor SB20358 alone nor its combination with PD098059 blocked the effect of IL-1ß on the expression of P2Y₂ receptor mRNA. Our results demonstrate that inflammatory mediators upregulate vascular P2Y₂ receptors at the transcriptional and at the functional level through protein kinase C and cyclooxygenase but not cAMP, extracellular signal–regulated kinases 1 and 2, or P38-dependent pathways. This may result in increased growth-stimulatory or contractile effects of extracellular UTP and ATP, which may be of importance in the development of vascular disease.

Key Words: P2Y₂ receptors • interleukin-1ß • competitive reverse transcription–polymerase chain reaction

	Introduction

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Extracellular nucleotides can be released in high concentrations in proximity to the vessel wall from endothelial cells during hypoxia and shear stress, from aggregating platelets, from inflammatory cells, and from vascular smooth muscle cells (VSMCs) when damaged.¹ ATP has been shown to be mitogenic for VSMCs.^{2 3 4} However, cloning of P2 receptors has revealed that several subtypes (P2Y₂, P2Y₄, and P2Y₆) are activated by pyrimidines (UTP and UDP). The first demonstration of extracellular UTP acting as a growth factor was performed in VSMCs.³ This has been confirmed and further studied in VSMCs and other cell types, such as renal mesangial cells, astrocytes, and osteoblasts.^{5 6 7 8 9} The mitogenic effect has been shown to be mediated by the UTP- and ATP-sensitive P2Y₂ receptor.¹⁰

The trophic effects may be of importance in the development of atherosclerosis and in neointimal formation after angioplasty.¹¹ In fact, after balloon angioplasty, stronger expression of the P2Y₂ receptor mRNA has been demonstrated in the neointima than in the media.¹² Furthermore, the P2Y₂ receptor is upregulated in the VSMC by growth factors and in the shift from the contractile to the synthetic phenotype.^{13 14} This change of phenotype is a prerequisite for the involvement of the VSMC in the development of atherosclerosis and neointima.¹¹ Interestingly, another P2 receptor subtype, the P2Y₁₂ receptor, which mediates ADP-stimulated platelet aggregation, has already been shown to be of clinical importance in atherosclerotic disease. The specific P2Y₁₂ receptor antagonists ticlopidine and clopidogrel prevent ischemic heart disease and stroke.¹⁵

Atherosclerosis is regarded as an inflammatory disease.¹⁶ Several pathophysiological studies in humans and animals have indicated the importance of cytokines in the development of atherosclerotic disease. Cytokines such as interleukin (IL)-1ß, interferon (IFN)-, and tumor necrosis factor (TNF)- can be released from accumulated macrophages and T lymphocytes, injured endothelium, and activated smooth muscle cells.^{11 16 17} Furthermore, aggregating platelets release ATP and UTP, which in turn may activate IL-1ß–converting enzyme to form bioactive IL-1ß and induce the release of IL-1ß from monocytes and macrophages.^{1 18 19} These cytokines can act as chemotactic factors for smooth muscle cells migrating into the intima, induce proliferation, modulate the smooth muscle cell phenotype, and regulate matrix breakdown and synthesis.^{20 21 22} The effects are due to IL-1ß–induced changes in constitutive and inducible gene expression or numbers of surface receptors.¹⁷

Whether inflammatory mediators affect vascular P2Y₂ receptor expression is unknown. Therefore, in the present study, we investigated the effects of inflammatory factors on P2Y₂ receptor transcription and characterized the involved intracellular signal transduction pathways.

	Methods

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Cell Culture
A culture of aortic smooth muscle cells was prepared essentially as described previously.³ Cells were passaged 4 times before use. Smooth muscle cells were identified by immunofluorescent staining of -actin filaments.

For time studies, the cells were kept in serum-free media for 48 hours, after which they were stimulated with IL-1ß (10 ng/mL) for 1, 6, and 24 hours. IL-1ß was introduced to the quiescent cells for 24 hours in the absence or presence of different signal pathway blockers added 1 hour before.

For DNA synthesis, the growth-arrested cells were incubated with IL-1ß and stimulated with UTP and ATP for 19 hours. The proliferation of cells was measured by using [³H]thymidine incorporation described previously.³

The measurement of cytosolic Ca²⁺ concentration was performed as described previously.¹³ Cells were stimulated with 100 µmol/L UTP, after which 10 µmol/L ionomycin was added as reference.

For cAMP, after 48 hours of starvation, the cells were incubated with forskolin for 5 minutes and IL-1ß for 5 and 45 minutes.²³ The reactions were terminated by ice-cold perchloric acid. After centrifugation, the supernatants were transferred to new tubes and neutralized with KOH. The cAMP contents were measured by using a cAMP assay kit according to the manufacturer’s instructions (Amersham). By the competition of binding protein between the labeled and unknown cAMP and by scintillation counting, the amounts of cAMP in the samples were determined from a linear standard curve.

Competitive RT-PCR
RNA was extracted from samples with the use of TRIzol reagent (GIBCO-BRL) by following the supplier’s instructions as described previously.¹³

Competitive reverse transcription (RT)–polymerase chain reaction (PCR) was carried out as described in detail previously.¹⁴ Specific primers for the rat P2Y₂ receptor²⁴ were designed (forward, 5'-ACCCGCACCCTCTATTACT-3'; reverse, 5'-CTTAGATACGA-TTCCCCAACT-3') generating a PCR product of 538 bp. A synthetic RNA competitor for the P2Y₂ receptor, bearing a deletion of 86 bp compared with the wild-type sequence, has previously been constructed.¹⁴ For competitive RT-PCR, 200 ng total RNA was mixed with different amounts of competitor RNA in 5 subsequent 1:5 dilution steps. RT-PCR was carried out by using the GeneAmp RNA PCR kit on a GeneAmp PCR system 2400 (Perkin-Elmer) as described previously.¹³ Because the studied P2Y₂ receptor is intronless within its coding region, PCR without the RT step was always used to exclude genomic DNA contamination.

A 10-µL PCR product was used for densitometric analysis by using NIH-Image software as described previously.¹³ Copy numbers were calculated and expressed as molecules per microgram total RNA.

Statistical Analyses
Values represent mean±SEM. Differences between groups were analyzed by the Student t test for unpaired analysis with the use of Statview. Differences were considered at P<0.05.

	Results

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Upregulation of P2Y₂ Receptor mRNA by Cytokines and LPS
IL-1ß increased the expression of P2Y₂ receptor mRNA in a time-dependent manner. The amount of P2Y₂ receptor mRNA had already increased after 6 hours of stimulation with IL-1ß (489±96% of control) and further increased after 24 hours of stimulation (Figure 1A). After 48 hours of stimulation, the levels of P2Y₂ receptor mRNA seemed to have reached a plateau. There was no significant difference between the cells stimulated with IL-1ß for 24 hours and those stimulated for 48 hours (723±17% and 774±310% of control, respectively). IL-1ß also dose-dependently increased the expression of P2Y₂ receptor mRNA, with the maximum effect at the concentration of 10 ng/mL (723±17% of control) and no further increase at 20 ng/mL (729±133% of control, Figure 1B). The effects of IFN- and TNF- on the expression of P2Y₂ receptor mRNA levels were also tested. Neither IFN- nor TNF- significantly increased the levels of P2Y₂ receptor mRNA (IFN-, 153±49% of control; TNF-, 123±24% of control; Figure 2). However, when either of them was coincubated with IL-1ß, it strongly enhanced the levels of P2Y₂ receptor mRNA by IL-1ß (IFN- or TNF- versus IL-1ß, 1000±46% or 1113±33% versus 559±52% of control). Bacterial lipopolysaccharide (LPS) alone also increased the levels of P2Y₂ receptor mRNA (223±61% of control, Figure 2).

View larger version (22K): [in this window] [in a new window]   Figure 1. A and B, Time-dependent (A) and dose-dependent (B) upregulation of P2Y2 receptor mRNA. Growth-arrested cells were incubated with IL-1ß for the indicated time or different concentrations (n=3). The amount of P2Y2 receptor mRNA was measured by competitive RT-PCR and presented as percentage of control. C, Gel electrophoresis of competitive RT-PCR for the detection of P2Y2 receptor mRNA levels. Growth-arrested cells were incubated without or with IL-1ß (10 ng/mL) for 24 hours. The amount of P2Y2 receptor mRNA was measured as described in Methods with the same concentration of total RNA together with 5 increasing concentrations of competitor standard in 1:5 dilution. Upper bands represent the wild-type product, and lower bands represent the competitor product. There is a clear shift in the equivalence point (arrows) in the IL-1ß–treated cells toward the right (higher concentration).

View larger version (13K): [in this window] [in a new window]   Figure 2. Effects of IL-1ß (10 ng/mL), IFN- (1000 U/mL), TNF- (10 ng/mL), and their combinations as well as LPS (1 µg/mL) on the expression of P2Y2 receptor mRNA (n=2 or 3). The amount of P2Y2 receptor mRNA was measured by competitive RT-PCR and presented as percentage of control.

IL-1ß Induced Increased Responses to UTP
IL-1ß alone had no effect on the accumulation of [³H]thymidine incorporation (10±5% of control). When IL-1ß was added together with UTP, DNA synthesis induced by UTP was significantly increased from 54±14% to 107±21% of control. Similar results were obtained when cells were stimulated with ATP combined with IL-1ß (228±30% versus 128±24% of control, Figure 3).

View larger version (12K): [in this window] [in a new window]   Figure 3. Effects of ATP (10 µmol/L) and UTP (10 µmol/L) in the presence or absence of IL-1ß (10 ng/mL) on the accumulation of [3H]thymidine incorporation expressed as percentage of control. Values represent mean±SEM from 10 or 11 experiments run in triplicate. *P<0.05 (Student t test, unpaired).

After incubation with UTP (100 µmol/L) for 48 hours to downregulate membrane receptors by internalization,²⁵ the cells were kept in serum-free medium for another 26 hours. After this, the response to 100 µmol/L UTP increased intracellular Ca²⁺ by 22±7% of reference (10 µmol/L ionomycin). The addition of IL-1ß (10 ng/mL) during the later 26 hours increased intracellular Ca²⁺ to a significantly higher extent, by 49±9% of reference (n=10 to 13).

Among P2Y receptors, both P2Y₂ and P2Y₄ receptors mediate the functions of UTP and ATP in rat aortic smooth muscle cells.^{26 27} To determine whether the increased response is mediated by the increased P2Y₄ receptors, we measured the levels of P2Y₄ receptor mRNA in the cells treated with IL-1ß under the same condition that we used for measuring P2Y₂ receptor mRNA. IL-1ß decreased the levels of P2Y₄ receptor mRNA (51±15% of control, n=3).

Transcriptional Regulation of P2Y₂ Receptor mRNA Levels by IL-1ß
In a previous study, we have shown that P2Y₂ receptor mRNA is rapidly degraded.¹³ To determine whether the upregulation of P2Y₂ receptor mRNA levels by IL-1ß involves transcriptional or posttranscriptional mechanisms, the RNA polymerase II inhibitor actinomycin D (5 µg/mL) was used. Actinomycin D significantly reduced the basal levels of P2Y₂ receptor mRNA expression (13±4% of control). In the presence of actinomycin D, IL-1ß (10 ng/mL) had no stimulatory effect on P2Y₂ receptor mRNA expression (12±5% of control, n=5).

Effects of Signal Pathway Blockers on Expression of P2Y₂ Receptor mRNA Induced by IL-1ß
Indomethacin,²³ a nonselective cyclooxygenase inhibitor, significantly inhibited the upregulation of P2Y₂ receptor mRNA expression induced by IL-1ß (Table). The selective protein kinase C (PKC) inhibitor RO-31-8220²⁸ also significantly inhibited the upregulation of P2Y₂ receptor mRNA by IL-1ß (Table). Neither indomethacin nor RO-31-8220 alone inhibited basal levels of P2Y₂ receptor mRNA expression (113±35% or 87±19% of control, n=2).

View this table: [in this window] [in a new window]   Table 1. Effects of Signal Pathway Blockers on IL-1ß–Induced Upregulation of P2Y2 Receptor mRNA Expression

Role of cAMP on Upregulation of P2Y₂ Receptor mRNA Expression by IL-1ß
cAMP is one of the second messengers activated by IL-1ß in other cell systems.²⁹ Forskolin (10 µmol/L), the activator of adenylate cyclase, significantly increased the expression of P2Y₂ receptor mRNA (259±65% of control, n=6). To determine whether cAMP is involved in the regulation of P2Y₂ receptor mRNA expression in rat aortic VSMCs by IL-1ß, we tested the effect of IL-1ß (10 ng/mL) on the accumulation of cAMP. After 5 minutes of stimulation, forskolin (10 µmol/L) significantly increased the amount of cAMP, whereas there was no increase in the cells stimulated with IL-1ß (262±29% and 105±10% of control, n=5 to 7). It was reported that IL-1ß reached the maximum effect on the accumulation of cAMP after 45 minutes²³ ; therefore, we prolonged the incubation time of IL-1ß. However, there was still no increase of cAMP (131±22% of control, n=6).

Lack of Involvement of ERK-1/2 and P38 MAPK in Upregulation of P2Y₂ Receptor mRNA Expression by IL-1ß
The signal pathways stimulated by IL-1ß may involve at least 3 types of mitogen-activated protein kinases (MAPKs): extracellular signal–regulated kinases 1 and 2 (ERK-1/2), P54 MAPK, and P38 MAPK.³⁰ To study which MAPK subtypes might be involved in the upregulation of P2Y₂ receptor mRNA expression by IL-1ß, the selective ERK-1/2 inhibitor PD098059 (30 µmol/L) and P38 MAPK inhibitor SB20358 (10 µmol/L) were used.^{13 31} PD098059 had no effect on the upregulation of P2Y₂ receptor mRNA by IL-1ß (312±83% versus 341±74% of control, n=7). SB20358 did not block IL-1ß–induced upregulation of P2Y₂ receptor mRNA expression (423±56% versus 450±44% of control, n=5). Furthermore, no inhibition was observed when the ERK-1/2 and P38 MAPK pathways were both blocked simultaneously by PD098059 and SB20358 (403±98% versus 460±73% of control, n=5).

	Discussion

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The main finding of the present study was that inflammatory mediators upregulate vascular P2Y₂ receptors at the transcriptional and the functional level. This may result in increased growth-stimulatory or contractile effects of extracellular UTP and ATP, which may be of importance in the development of vascular disease.

The effects of inflammatory mediators on P2Y₂ receptor mRNA were measured with a quantitative competitive RT-PCR recently developed in our laboratory.¹⁴ A crucial point in competitive PCR methods is to keep the difference between standard and sample as small as possible. The method used in the present study is based on an artificial RNA standard yielding a deletion of 86 bp. The inclusion of the shorter RNA competitor of known concentrations generated distinguishable signals from the sample while controlling all steps of the reaction, including the RT step. The method has been characterized in detail previously and found to have low intra-assay variation and high sensitivity.¹⁴

IL-1ß significantly increased the levels of P2Y₂ receptor mRNA by >7-fold. The bacterial endotoxin LPS also upregulated the P2Y₂ receptor mRNA expression, although to a lesser extent than IL-1ß. IFN- and TNF- did not increase P2Y₂ receptor mRNA levels, per se, but dramatically enhanced the effect of IL-1ß on the expression of P2Y₂ receptor mRNA in a synergistic way. IL-1 receptors have been divided into 2 subtypes: IL-1RI, which is responsible for the biologic effects of IL and shows low levels on all cells, and IL-1RII, which serves as a decoy receptor. IFN- or TNF- may act synergistically with IL-1ß by increasing the number of IL-1RI.²⁹

Other investigators have shown that the upregulation of bradykinin B₁ receptor mRNA and of vascular endothelial growth factor mRNA expression by IL-1ß is due to the increased transcriptional rate and stability of mRNA.^{32 33} Because P2Y₂ receptor mRNA is rapidly degraded, with a half-life of 1 hour,¹³ increased stability could be involved. However, in the presence of the mRNA polymerase II inhibitor actinomycin D, IL-1ß did not induce any increase at mRNA levels, indicating that the effect of IL-1ß is mediated through de novo synthesis of P2Y₂ receptor mRNA and not through inhibition of the degradation rates for P2Y₂ mRNA.

The transcriptional upregulation was paralleled with increased functional effects, because IL-1ß enhanced the UTP-stimulated accumulation of [³H]thymidine incorporation, which was further confirmed by increased release of intracellular Ca²⁺ concentration in IL-1ß–treated cells. IL-1ß also significantly enhanced the UTP-stimulated release of intracellular Ca²⁺ in desensitized cells, indicating that the P2Y₂ receptor number on the cell surface was increased. This is probably the result of de novo protein synthesis, inasmuch as it has been demonstrated that after desensitization with long-term incubation of the P2Y₂ receptor agonist UTP, the recovery of cell surface receptors is dependent on de novo protein synthesis and not the reexpression of internalized receptors.²⁵ Because IL-1ß decreased the mRNA levels for the P2Y₄ receptor,²⁶ it seems unlikely that the P2Y₄ receptor has contributed to the increased functional responses to UTP induced by IL-1ß.

Some effects of IL-1ß have been shown to be dependent on the production of intracellular prostaglandins.¹⁷ This intracellular pathway seems important for the upregulation of P2Y₂ receptor mRNA expression by IL-1ß because the nonselective cyclooxygenase inhibitor indomethacin inhibited the effect of IL-1ß on the expression of P2Y₂ receptor mRNA. During smooth muscle cell phenotype change from the contractile to the synthetic phenotype, P2Y₂ receptor mRNA was upregulated.¹⁴ Our findings support the opinion of Pomerantz and Hajjar³⁴ that prostaglandins play an important role in the modulation of arterial smooth muscle cell differentiation from contractile to synthetic phenotype. This can also be regarded as one of the reasons why TNF- dramatically enhances the upregulation of P2Y₂ receptor mRNA by IL-1ß. TNF- and IL-1ß synergistically stimulate prostaglandin production.³⁵ At the concentration of 10 µmol/L, indomethacin cannot block the IL-1ß–induced release of HETEs, arachidonic acid lipoxygenase metabolites, which activate ERK-1/2 and Jun N-terminal kinase to modulate the functions of VSMCs.^{36 37 38 39} This may be one of the explanations of why indomethacin significantly (but only by 59%) blocked the effect of IL-1ß on the expression of P2Y₂ receptor mRNA.

The selective PKC inhibitor RO-31-8220 significantly inhibited the upregulation of P2Y₂ receptor mRNA expression, which is in agreement with previous findings that activation of PKC is a part of the IL-1ß signal mechanisms.²⁹ At a concentration of 0.1 µmol/L, RO-31-8220 inhibits not only PKC but also MAPK activated protein kinase-1ß and p70 S6 kinase.⁴⁰ Lack of inhibition of PD098059 or rapamycin (100 nmol/L) on the accumulation of P2Y₂ receptor mRNA expression suggests that the inhibitory effect of RO-31-8220 on the upregulation of P2Y₂ receptor mRNA is mediated by the inhibition of PKC but not by the inhibition of MAPK kinase-1 kinase and p70 S6 kinase.

Prostaglandins may act as upstream activators of cAMP.¹¹ As a second messenger, cAMP induces the upregulation of bradykinin B₂ receptor mRNA and ß₂-adrenergic receptor mRNA expression.^{23 41} On human myeloid leukocytes, cAMP can induce the transient upregulation of P2Y₂ receptor mRNA.⁴² In agreement with these findings, we found that forskolin increased the expression of P2Y₂ receptor mRNA. It was also reported that IL-1ß stimulated the accumulation of cAMP on rat VSMCs.⁴³ In contrast, IL-1ß did not stimulate cAMP production in our rat aortic VSMCs after either 5 or 45 minutes, indicating that cAMP is not involved in the upregulation of P2Y₂ receptor mRNA expression by IL-1ß. This is consistent with another investigation in which prostaglandin upregulates insulin-like growth factor binding protein-3 expression via cAMP-independent pathways.⁴⁴

The signal pathways induced by cytokines may involve 3 subtypes of MAPKs: P42 and P44 MAPKs, Jun kinase/stress-activated protein kinase, and P38 MAPK.³⁰ It is possible that these 3 subtypes play different roles in the regulation of gene expression by IL-1ß. One study showed that the upregulation of LDL receptor expression requires the activation of ERK-1/2 but not P38, which suppresses the receptor expression.⁴⁵ In contrast, another study indicated that IL-1ß via ERK-1/2 activation downregulated -platelet-derived growth factor receptor expression.⁴⁶ It was also reported that gene expression required the simultaneous activation of ERK-1/2 and P38 MAPK.⁴⁷ Our previous study has shown that activation of ERK-1/2 is necessary for FCS-induced upregulation of P2Y₂ receptor mRNA.¹³ However, the finding in the present study is that none of PD098059, SB20358, or their combination significantly inhibits the upregulation of P2Y₂ receptor mRNA expression by IL-1ß. This excludes ERK-1/2 and P38 MAPK pathways in the regulation of P2Y₂ receptor mRNA expression by IL-1ß, but it is possible that a Jun kinase/stress-activated protein kinase pathway is involved, which was found to be involved in the induction of NO synthase II by IL-1ß.⁴⁸ Although we have not directly confirmed the specific inhibitory effects of the different blockers of the intracellular signal pathways, the concentrations that we used were based on previous studies in similar cell systems in which they have been shown to be selective.

In summary, IL-1ß and other inflammatory mediators induce transcriptional upregulation of the P2Y₂ receptor in rat aortic smooth muscle cells, resulting in increased UTP-stimulated DNA synthesis and intracellular Ca²⁺ release. The signal pathways are PKC and cyclooxygenase dependent but are not mediated via cAMP–, ERK-1/2–, or P38 MAPK–dependent pathways. It is possible that this may result in an increase in UTP- and ATP-stimulated proliferation of VSMCs in atherosclerosis and in neointimal formation after balloon angioplasty.

	Acknowledgments

 
This study has been supported by the Swedish Heart and Lung Foundation, Swedish Medical Research Council grants 13130 (D.E.) and 5958 (L.E.), the Tore Nilsson Foundation, the Jeansson Foundation, and the Swedish Hypertension Society.

Received February 29, 2000; accepted May 24, 2000.

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