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(Circulation. 2004;110:1128-1133.)
© 2004 American Heart Association, Inc.

Original Articles

Laminar Flow Activates Peroxisome Proliferator-Activated Receptor- in Vascular Endothelial Cells

Yi Liu, PhD; Yi Zhu, MD; Francois Rannou, MD PhD; Tzong-Shyuan Lee, DVM PhD; Kitty Formentin, PhD; Lingfang Zeng, PhD; Xiaohui Yuan, PhD; Nanping Wang, MD; Shu Chien, MD PhD; Barry M. Forman, MD PhD; John Y.-J. Shyy, PhD

From the Division of Biomedical Sciences, University of California at Riverside (Y.L., Y.Z., F.R., T.-S.L., K.F., L.Z., J.Y.-J.S.); Department of Bioengineering and Whitaker Institute of Biomedical Engineering, University of California at San Diego, La Jolla (N.W., S.C.); and Division of Molecular Medicine, City of Hope National Medical Center, Duarte, Calif (X.Y., B.M.F.).

Correspondence to Dr John Y. Shyy, Division of Biomedical Sciences, University of California at Riverside, Riverside, CA 92521-0121. E-mail john.shyy{at}ucr.edu

Received November 6, 2003; de novo received January 28, 2004; revision received March 30, 2004; accepted April 9, 2004.

	Abstract

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Background— Steady laminar flow is atheroprotective, in part because of its antiinflammatory effects on vascular endothelial cells (ECs). We studied the activation of peroxisome proliferator-activated receptor- (PPAR) in ECs in response to laminar flow and the associated antiinflammatory effect.

Methods and Results— Using flow channel with cultured ECs, we found that laminar flow activated the PPAR-mediated PPAR-responsive element (PPRE) activity and increased the mRNA encoding CD36, a PPAR-targeted gene. Analysis of the CD36 promoter revealed that PPRE was required for flow activation. Laminar flow induced the GAL-PPAR-LBD fusion protein, which suggests that flow activation of PPAR was ligand dependent. The pharmaceutical inhibitors of phospholipase A2 (PLA2) and cytochrome P450 epoxygenases (CYP450s) were able to block the laminar flow-activated PPAR. We also showed that lipid extracts from flow media contained ligands for the activation of PPAR in other cell types. This paracrine activation exerted antiinflammatory effects in ECs and THP-1 cells, including the suppression of cytokine-induced nuclear factor-B activation and expression of intercellular adhesion molecule-1.

Conclusions— Laminar flow activates endogenous PPAR in ECs, which is ligand dependent. The flow production of PPAR ligands is through the PLA2-CYP450 pathway, and the induced PPAR ligands exert antiinflammatory effects in several types of cells.

Key Words: cells, endothelial • receptors, peroxisome proliferator-activated • inflammation

	Introduction

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Depending on the geometry of the arterial tree, blood flow in the straight part of vessels is steady and laminar, whereas that in the bends and branches is disturbed.¹ The focal distribution of atherosclerotic lesions in the disturbed flow areas indicates the pivotal role of local flow in atherogenesis.² Because early atherosclerotic events involve the inflammatory responses of vascular endothelial cells (ECs), disturbed flow patterns with high shear stress gradients are considered to be proinflammatory, whereas steady laminar flow is antiinflammatory.^3,4 Using flow channel systems, we and others demonstrated that the application of flow to cultured ECs transiently induced genes encoding chemoattractants, adhesion molecules, and cytokines, which is due in part to the activation of nuclear factor-B (NF-B) and AP-1.^5,6 The induction of these genes has been suggested to be the endothelial response to the rapid change of shear stress (/t).⁷ In agreement with this hypothesis, prolonged laminar flow suppresses the expression of proinflammatory molecules.⁸ To date, the molecular basis underlying the antiinflammatory effect of laminar flow is still elusive.

Peroxisome proliferator-activated receptor (PPAR) -, -ß/, and - constitute a subfamily of nuclear receptors. Among PPARs, PPAR is largely expressed in monocytes/macrophages, adipocytes, and intestinal cells in which PPAR regulates genes involved in cell differentiation and lipid uptake and storage (eg, aP2, CD36).⁹ The ligand-binding domain (LBD) of PPAR can bind a wide range of ligands that have distinct structures,¹⁰ including the natural ligand 15-deoxy-^12,14-prostaglandin J₂ (15d-PGJ₂) and synthetic ligand thiozolidinediones (TZDs). PPAR is also present in ECs and vascular smooth muscle cells and seems to be related to pathophysiological process such as atherosclerosis.^11,12 Feeding apolipoprotein E-deficient mice with synthetic TZDs such as troglitazone reduced the homing of labeled monocytes/macrophages to atherosclerotic plaque.¹³ Similarly, troglitazone attenuated lesion formation and monocyte/macrophage recruitment in mice deficient in LDL receptor.¹⁴ Activation of PPAR by TZDs or overexpression of a constitutively active mutant of PPAR in cultured ECs significantly reduced the expression of vascular adhesion molecule-1 (VCAM-1) and endothelin-1^13,15 because of the attenuation of NF-B and/or AP-1.¹⁵

Given the antiinflammatory effect of PPAR and the role of shear stress in EC biology, we investigated whether laminar flow modulates PPAR in ECs, and, if it does, the functional consequence in terms of antiinflammatory effect.

	Methods

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Cell Culture and Flow Experiments
Bovine aortic endothelial cells (BAECs), human umbilical vein endothelial cells (HUVECs), monocytic cell line THP-1, and CV-1 cells were cultured under standard culture conditions. The flow experiments were performed as previously described.¹⁶ In brief, a parallel-plate flow channel was used to impose a laminar shear stress of 12 dyne/cm² on a confluent monolayer of cells seeded on glass slides. The flow system was kept at 37°C and ventilated with 95% humidified air with 5% CO₂.

Plasmids, Small Interfering RNA, and Transient Transfection
PPREx3-TK-Luc, MH100x4-TK-Luc, GAL-mPPAR-LBD, GAL-retinoid X receptor (hRXR)-LBD, CMX-mPPAR, CMX-mPPAR-L466/467A, CD36-273-Luc, and CD36-261-Luc were described previously.^17,18 GAL-mPPAR-LBD-L328A, containing the indicated point mutation, has normal heterodimerization and DNA-binding properties but diminished affinity for ligands. Cells were also transfected with CMV-ß-gal for transfection control. The various plasmids were transiently transfected into cells by the use of lipofectamine (Invitrogen).

The small interfering RNA (siRNA) nucleotide sequences for human PPAR1 and control pGL3 are as follows: 5'-AAUGGAAGACCACUCCCACUC-3' and 5'-CUUACGCUGAGUACUUCGA-3', respectively. For siRNA transfection, HUVECs in a 6-well plate with 70% confluence were transfected with double-strand siRNAs at a final concentration of 300 nmol/L by the use of oligofectamine (Invitrogen).

Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated from cells with the TRIzol reagent (Invitrogen), and the isolated RNA was converted into cDNA by use of reverse transcriptase with oligo(dT) as the primer. The obtained cDNAs and various primers were then added to the Brilliant SYBR Green QPCR Master Mix (Stratagene) for quantitative polymerase chain reaction (PCR).

Lipid Extraction
The conditioned media collected from flow experiments were mixed with 7 volumes of CHCl₃/MeOH (2:1) containing 0.01% butylated hydroxytoluene. The mixtures were vortexed and centrifuged at 2000 rpm for 10 minutes. The lower chloroform layer was collected and dried under nitrogen at 25°C. The dried lipids were reconstituted in 10% FBS DMEM media to the original volumes.

Immunoblotting Analysis
Cell lysates were resolved by 10% SDS-PAGE and transferred to a nitrocellulose membrane, and the IB protein was detected with a rabbit anti-IB polyclonal antibody (Pharmingen). The bound primary antibody was recognized with a goat anti-rabbit IgG-horseradish peroxidase conjugate (Santa Cruz Biotechnology) and visualized by the ECL detection system (Amersham).

Statistical Analyses
The results were expressed as mean±SD from at least 3 independent experiments. The data were analyzed by 2-tailed Student t test. Probability values of <0.05 were considered statistically significant.

	Results

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Laminar Flow Activates the PPAR-Mediated Transcription
To examine the effect of flow on the PPAR-mediated transcription, we applied a laminar flow with shear stress at 12 dyne/cm² to BAECs transiently transfected with a luciferase reporter driven by 3 copies of PPAR responsive element (PPRE) (ie, PPREx3-TK-Luc). As shown in Figure 1A, laminar flow induced 4.2±0.4 times luciferase activity compared with static controls. As a control, treating BAECs with rosiglitazone, a PPAR ligand, under static conditions caused 4.3±0.2 times induction. Because PPREx3-TK-Luc responds to all PPAR isoforms,^17,19 we included bisphenol A diglycide ether (BADGE), a PPAR-specific antagonist, in the perfusing media to explore whether PPAR is involved in the flow-activated PPRE. BADGE greatly reduced the induction of PPREx3-TK-Luc by flow. To further confirm that the flow induction of PPRE is mediated through PPAR activation, we attempted to block the endogenous PPAR with CMX-mPPAR-L466/467A encoding a dominant negative mutant of PPAR.²⁰ As shown in Figure 1B, the flow-induced PPREx3-TK-Luc was drastically reduced by the expression of mPPAR-L466/467A compared with cells transfected with CMX-mPPAR, which encodes the wild-type PPAR.

View larger version (25K): [in this window] [in a new window]   Figure 1. Laminar flow upregulates PPAR-mediated transcription in ECs. A, BAECs were transfected with PPREx3-TK-Luc together with CMV-ß-gal. After becoming confluent, the transfected cells were kept as static controls, subjected to a laminar flow at 12 dyne/cm2, or stimulated with rosiglitazone (10 µmol/L) in the presence or absence of BADGE (100 µmol/L) for 8 hours. B, BAECs were cotransfected with PPREx3-TK-Luc and CMX-mPPAR or CMX-mPPAR-L466/467A. The transfected cells were kept as static controls, subjected to a laminar flow, or stimulated with rosiglitazone for 8 hours. Cells were then lysed for luciferase and ß-gal activity assays. The luciferase activity was normalized to that of ß-gal. The bars represent the relative luciferase activity, defined as the normalized luciferase activity of various experiments in reference to that of static controls set as 1. All results are mean±SD from 3 separate sets of experiments. DMSO indicates dimethyl sulfoxide. *P<0.05.

Because CD36 is a PPAR target gene,²¹ we performed quantitative reverse transcription (RT)-PCR and found that flow increased CD36 mRNA by 2.8±0.4 times, which was comparable to the augmentation by rosiglitazone (Figure 2A). We investigated further the role of PPRE in flow induction of the CD36 promoter. As shown in Figure 2B, flow increased the expression of luciferase controlled by the CD36 promoter (ie, CD36-273-Luc). Deletion of PPRE (ie, CD36-261-Luc) abolished the induction by flow or rosiglitazone.

View larger version (24K): [in this window] [in a new window]   Figure 2. Laminar flow induction of CD36 is PPAR/PPRE dependent. A, Total RNAs were isolated from static HUVECs or those subjected to laminar flow or rosiglitazone stimulation for 24 hours. The level of CD36 mRNA was determined by quantitative RT-PCR with ß-actin as an internal control. The relative CD36 mRNA is defined as the mRNA level of CD36 in cells exposed to various stimuli in reference to that of static controls set as 1. B, BAECs transfected with CD36-273-Luc or CD36-261-Luc were kept as static controls, subjected to laminar flow, or stimulated with rosiglitazone for 8 hours. The cells were then lysed for luciferase and ß-gal activity assays. The bars represent the relative luciferase activity, defined as the normalized luciferase activity of various experiments in reference to that of static controls. *P<0.05.

Laminar Flow Activation of PPAR Is Mediated Through the LBD
To examine the ligand dependence of flow activation of PPAR, we transfected BAECs with a GAL4 reporter construct (ie, MH100x4-TK-Luc) and a vector expressing GAL-mPPAR-LBD fusion protein in which the DNA binding domain of GAL4 is linked to the LBD of the mouse PPAR.¹⁷ The transfected cells were kept as static controls, subjected to laminar flow, or treated with rosiglitazone. Compared with static controls, flow and rosiglitazone increased the luciferase activity by 13.9±2.1 and 35.3±3.3 times, respectively (Figure 3A). A mutant construct (ie, GAL-mPPAR-LBD-L328A), in which the Leu-328 within the LBD was replaced by an Ala or the LBD of RXR (ie, GAL-hRXR-LBD), did not respond to flow. These results suggest that the flow activation of PPAR was through a ligand-dependent mechanism.

View larger version (27K): [in this window] [in a new window]   Figure 3. Laminar flow activation of PPAR is ligand dependent, and the ligand(s) are released into the perfusing media. A, BAECs cotransfected with MH100x4-TK-Luc and GAL-mPPAR-LBD, GAL-mPPAR-LBD-L328A, or GAL-hRXR-LBD were kept under static conditions or subjected to laminar flow or rosiglitazone for 8 hours. B, CV-1 cotransfected with MH100x4-TK-Luc and GAL-mPPAR-LBD or GAL-mPPAR-LBD-L328A was incubated with conditioned media collected from static or flow experiments or with lipid extracts from the conditioned media. Cells were also incubated with media or the lipid extracts from mock shearing experiments that had no cells in the flow channel. C, BAECs transfected with PPREx3-TK-Luc were treated with conditioned media, lipid extracts collected from static or sheared cells, or rosiglitazone. All cells were lysed for luciferase and ß-gal activity assays. The results represent the relative luciferase activity, defined as the normalized luciferase activity of various experiments in reference to that of static controls in A and static media in B and C. *P<0.05.

The flow activation of the LBD of PPAR could be due to either intracellular ligand(s) acting via an intracrine mechanism or those released to the perfusing media in an autocrine or paracrine manner. To investigate whether flow can cause the release of PPAR ligands, CV-1 cells transfected with MH100x4-TK-Luc and GAL-mPPAR-LBD were incubated with conditioned media collected from static or flow experiments. As shown in Figure 3B, the incubation of flow media increased the luciferase activity by 7.5±1.1 times, compared with media from static cultures. In parallel controls, flow media involving no ECs (mock shearing) had little effect on GAL-mPPAR-LBD. To delineate whether lipid(s) is (are) the active component causing PPAR activation, we extracted the flow media with CHCl₃/MeOH. Incubation of such lipid extracts caused a comparable increase (5.8±1.9 times) in luciferase activity. However, only basal luciferase activity was detected in cells transfected with GAL-mPPAR-LBD-L328A in response to lipid extracts from static, flow, or mock shearing media or rosiglitazone. The lipid extracts from flow media also induced PPREx3-TK-Luc in ECs never exposed to flow (Figure 3C), indicating that the active ingredients in lipid extracts can activate the endogenous PPAR.

PLA2-CYP450 Pathway Is Involved in PPAR Activation in Response to Laminar Flow
We included bromoenol lactone (BEL), a phospholipase A2 (PLA2) inhibitor, in flow experiments to investigate whether PLA2 was involved in the EC production of PPAR ligands. As shown in Figure 4A, BEL drastically reduced the flow induction of GAL-mPPAR-LBD in BAECs. This result suggests the involvement of PLA2 and its product, arachidonic acid (AA), in flow-producing PPAR ligands. Because AA can be further metabolized by cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochrome P450 epoxygenases (CYP450s), we used indomethacin, nordihydroguaiaretic acid (NDGA), and 1-aminobenzotriazole (1-ABT), the respective inhibitors for the 3 enzymes, to determine which of the 3 pathways produces PPAR ligands. As shown in Figure 4B, 1-ABT was able to reduce the flow-induced GAL-mPPAR-LBD, which was dose dependent. On the contrary, indomethacin and NDGA had no inhibitory effect. The inhibition by BEL and 1-ABT suggested that the PLA2-CYP450 pathway mediates the flow activation of PPAR in ECs. Furthermore, BEL and 1-ABT showed a similar inhibitory effect on the flow-induced PPREx3-TK-Luc in BAECs (Figure 4C).

View larger version (29K): [in this window] [in a new window]   Figure 4. The PLA2-CYP450 pathway is involved in the flow activation of PPAR. In A and B, BAECs transfected with MH100x4-Luc and GAL-mPPAR-LBD were kept as static controls, subjected to a laminar flow, or stimulated with rosiglitazone in the presence or absence of BEL (5 µmol/L) (A) or 1-ABT (1, 5 mmol/L), indomethacin (5, 10 µmol/L), or NDGA (5, 10 µmol/L) (B) for 8 hours. C, BAECs transfected with PPREx3-TK-Luc were kept as static controls, subjected to flow or rosiglitazone in the presence or absence of BEL or 1-ABT. The bars represent the relative luciferase activity, defined as the normalized luciferase activity of various experiments in reference to that of static cells treated with dimethyl sulfoxide (DMSO). *P<0.05.

Flow Media Exhibit Antiinflammatory Effect
Because of the paracrine effect of flow media on PPAR activation (Figure 3B, 3C) and the antiinflammatory effect of PPAR,¹⁵ we tested whether the lipid extracts of flow media could inhibit intercellular adhesion molecule-1 (ICAM-1) induced by the inflammatory cytokine interleukin-1ß (IL-1ß). Quantitative RT-PCR revealed that IL-1ß increased the expression of ICAM-1 mRNA in HUVECs pretreated with lipid extracts from static media (Figure 5A). Such an induction was greatly reduced by the pretreatment of lipid extracts from perfusing media or 15d-PGJ₂. Because the antiinflammatory effect of 15d-PGJ₂ is largely due to the receptor-independent mechanism,²² we used the PPAR-specific siRNA to suppress the expression of PPAR and to test whether PPAR is involved in the antiinflammatory effect of perfusing media. Transfection of HUVECs with siRNA reduced the expression of PPAR mRNA by 60%. This reduction of PPAR reversed the inhibitory effect of lipid extracts on the IL-1ß-induced ICAM-1 mRNA (Figure 5A). In contrast, PPAR siRNA had a marginal effect on the 15d-PGJ₂-suppressed ICAM-1 mRNA. We also tested the antiinflammatory effect of perfusing media on cells other than ECs. Lipopolysaccharide (LPS) treatment of monocytes increases the production of NO.^23,24 Preincubation of these cells with lipid extracts from perfusing media also inhibited such an induction of NO (Data Supplement Figure I).

View larger version (37K): [in this window] [in a new window]   Figure 5. PPAR contributes to the antiinflammatory effect of the flow media. A, HUVECs were transfected with PPAR or pGL3 siRNAs as indicated. The transfected cells were then treated with the lipid extracts from the conditioned media collected from the static or flow experiments or 15d-PGJ2 (5 µmol/L) for 1 hour. IL-1ß was then added at a concentration of 5 ng/mL and incubated for another 2 hours. The total RNAs were isolated, and the levels of the ICAM-1 and PPAR mRNA were determined by quantitative RT-PCR. The fold of induction is defined as the mRNA level compared with that of static controls. B, HUVECs were pretreated with the lipid extracts from the static media (lanes 1 to 4), flow media (lanes 5 to 8), or 15d-PGJ2 (lanes 9 to 12) in the presence or absence of BADGE (100 µmol/L) for 1 hour. LPS was then added to the HUVECs at a concentration of 1 µg/mL and incubated further for 30 minutes. The collected cell lysates were immunoblotted with anti-IB antibody. *P<0.05.

A major antiinflammatory effect of PPAR is its inhibition of NF-B-regulated transcription.¹⁵ We determined whether lipid extracts from flow media would inhibit the IB degradation in HUVECs stimulated with LPS. Immunoblotting results in Figure 5B show that the LPS-induced IB degradation was significantly suppressed by the lipid extracts (lane 2 versus 6). However, the inclusion of BADGE resulted in a partial degradation of IB (lane 6 versus 8), which suggested that PPAR was involved in the flow media inhibition of NF-B. Consistent with these results, the lipid extracts also inhibited the NF-B-mediated luciferase induction (Data Supplement Figure II).

	Discussion

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The application of flow to ECs cultured in a flow channel causes the transient activation of many genes involved in inflammation. Prolonged laminar flow protects ECs by downregulating these molecules.⁸ The antiinflammatory effect of laminar flow is evidenced by the finding that the preexposure of ECs to laminar flow inhibits IL-1ß- or tumor necrosis factor--induced NF-B activation, VCAM-1 expression, and monocyte adhesion.²⁵ Because of the antiinflammatory role of PPAR in ECs, we studied the effect of laminar flow on the activation of PPAR. The major findings are that (1) flow activates the endogenous PPAR in ECs, leading to the increased expression of the PPAR target gene; (2) flow produces PPAR ligands through the PLA2-CYP450 pathway; and (3) the induced PPAR ligands exert antiinflammatory effects.

Most of the natural compounds binding to PPAR are the metabolites derived from AA. Further study of the PPAR ligands generated by flow suggested that the AA metabolites could be the active components activating PPAR in ECs. Intracellular AA is mainly esterified to glycerophospholipids on the cell membrane. Activation of phospholipases (eg, cytosolic PLA2) releases AA from the phospholipid pools. The free AA is quickly converted to oxidative metabolites through 3 enzymatic pathways: (1) prostaglandins, thromboxane, and prostacyclin by COXs; (2) leukotrienes, hydroxyeicosatetraenoic acids (HETEs), and lipoxins by LOXs; and (3) epoxyeicosatrienoic acids and -terminal HETEs by CYP450s.²⁶ As a potent PPAR ligand, 15d-PGJ₂ is generated by the COX-prostaglandin H₂ synthase-prostaglandin D₂ synthase pathway.²⁷ Laminar flow has been shown to activate PLA2, leading to AA release,²⁸ and to induce the expression of COX2 and lipocalin-type prostaglandin D₂ synthase in ECs.²⁹ Thus, it seems that laminar flow also produces 15d-PGJ₂-like molecules to activate PPAR by activating the COX pathway. However, indomethacin and NDGA had a marginal inhibitory effect (Figure 4B); even the concentrations of these 2 inhibitors were much higher than their IC₅₀.^30,31 The inhibition of the flow-induced GAL-mPPAR-LBD and PPREx3-TK-Luc by 1-ABT suggests that the activation and ligand production of PPAR by laminar flow are mediated through CYP450s but not COXs or LOXs. Although 15d-PGJ₂ is a potent PPAR ligand, its affinity for PPAR is low (EC₅₀ 2 µmol/L).¹⁷ The level of 15d-PGJ₂ in vivo is several orders of magnitude lower than that required to activate PPAR in vitro.³² Taken together, it is likely that the metabolic products of CYP450s rather than those of COXs are the physiological ligands for PPAR released by ECs in response to laminar flow. More than 50 CYP450 genes belonging to 14 families in mammalian cells have been identified. Among them, several, including CYP1A1, 1B1, 2C8, and 2J2, are expressed in ECs.³³ High throughput DNA microarray screening has revealed that laminar flow upregulates CYP1A1 and 1B1 in ECs,^34,35 which suggests that laminar flow activates CYP450. However, PLA2-CYP450-independent pathways may also exist to activate PPAR. This possibility is in agreement with the partial inhibition of the flow-activated GAL-PPAR-LBD by the PLA2 or CYP450 inhibitors.

The discrepant effect of siRNA on the flow media- versus 15d-PGJ₂-suppressed ICAM-1 mRNA indicates that the antiinflammatory effect of flow media is largely due to PPAR but not 15d-PGJ₂ or its analogues. Laminar flow also activates several other antiinflammatory proteins, including HO-1.³⁶ It is not known whether those induced molecules exert any paracrine effect on the neighboring cells. However, we showed that the lipid extracts from perfusing media were able to suppress the cytokine-induced inflammatory responses in other cell types such as monocytes. This antiinflammatory effect seems to be due to the inhibition of IB degradation and the ensuing inactivation of NF-B.

	Acknowledgments

 
This study was supported in part by NIH grant HL56707. We thank Dr Thomas M. McIntyre, University of Utah, for providing CD36-273-Luc and CD36-261-Luc. Dr Ying Zhu and Andre Morgan are appreciated for their technical assistance.

	Footnotes

 
The online-only Data Supplement, which contains additional figures, is available with this article at http://www.circulationaha.org.

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