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(Circulation. 2005;111:2227-2232.)
© 2005 American Heart Association, Inc.

Vascular Medicine

Endothelium-Intrinsic Requirement for Hif-2 During Vascular Development

Li-Juan Duan, MSc; Yahui Zhang-Benoit, MSc; Guo-Hua Fong, PhD

From the Center for Vascular Biology, Departments of Cell Biology and Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Conn.

Reprint requests to Guo-Hua Fong, PhD, Center for Vascular Biology, University of Connecticut Health Center, 263 Farmington Ave, Farmington, CT 06030-3501. E-mail fong{at}nso2.uchc.edu

Received May 17, 2004; revision received December 11, 2004; accepted December 17, 2004.

	Abstract

Top Abstract Introduction Methods Results and Discussion References

 
Background— The development of the vascular system is a complex process that involves communications among multiple cell types. As such, it is important to understand whether a specific gene regulates vascular development directly from within the vascular system or indirectly from nonvascular cells. Hypoxia-inducible factor-2 (Hif-2, or endothelial PAS protein-1 [EPAS-1]) is required for vascular development in mice, but it is not clear whether its requirement resides directly in endothelial cells.

Methods and Results— To address this issue, we expressed Hif-2 cDNA in the vascular endothelium of Hif-2^–/– embryos by an embryonic stem (ES) cell–mediated transgenic approach and assessed whether endothelium-specific reexpression of Hif-2 could rescue vascular development. Here we report that although ES cell–derived Hif-2^–/– embryos developed severe vascular defects by embryonic day (E) 11.5 and died in utero before E12.5, endothelium-specific expression of Hif-2 cDNA restored normal vascular development at all stages examined (up to E14.5) and allowed Hif-2^–/– embryos to survive at a frequency comparable to that of Hif-2^+/– embryos. Furthermore, we found that Tie-2 expression was significantly reduced in Hif-2^–/– mutants but was restored by Hif-2 cDNA expression.

Conclusions— These data demonstrate an intrinsic requirement for Hif-2 by endothelial cells and imply that hypoxia may control endothelial functions directly via Hif-2–regulated Tie-2 expression.

Key Words: angiogenesis • morphogenesis • hypoxia • endothelium

	Introduction

Top Abstract Introduction Methods Results and Discussion References

 
Soon after gastrulation, the rapid expansion of embryonic tissues leads to an increased rate of oxygen consumption and a decreased rate of oxygen diffusion, thus creating a partially hypoxic environment. The embryo responds to such an environmental challenge by developing a circulatory system to deliver nutrition and oxygen and to remove metabolic waste products. Thus, hypoxia serves as the primary stimulator of vascular development by triggering the expression of angiogenic factors such as vascular endothelial growth factor-A.^1–5

Two critical mediators in the hypoxia response pathway are hypoxia-inducible factor-1 (HIF-1)^4,6–8 and hypoxia-inducible factor-2 (HIF-2; Hif-1 and Hif-2 in mice, respectively). HIFs are heterodimeric complexes between - and ß-subunits.^9,10 Both HIF-1 and HIF-2 are transcription factors that contain a basic helix-loop-helix motif, a PAS domain, transcription-activation domains, and oxygen-dependent degradation domains.^8,9,11 Although the ß-subunit is expressed constitutively, both HIF-1 and HIF-2 are regulated by the ubiquitylation/proteosome degradation pathway.^12–15 Under normoxic conditions, a specific proline residue in each oxygen-dependent degradation domain is hydroxylated by HIF-specific proline hydroxylases (PHDs/HPHs).^13,15,16 Hydroxylated HIF-1 and HIF-2 bind to von Hippel Lindau (VHL) protein, a component of the E3 ubiquitin-ligase complex. Such binding interactions direct HIF-1 and HIF-2 for ubiquitylation and subsequent degradation. Under hypoxic conditions, proline-specific hydroxylation does not occur, and therefore, HIF -subunits accumulate.^13,15,16

Studies have shown that mouse Hif genes are important for vascular development.^8,17–22 Hif-2 also plays other roles in mouse development and physiology, including homeostasis regulation,^23,24 apoptosis,²⁵ lung development,²⁶ and a multiorgan function in adult mice.²⁴ These studies also demonstrated that Hif-2 is expressed in multiple cell lineages. Such complex roles of Hif-2 prompted us to examine whether its role in vascular development was a direct one or an indirect derivative of its functions in other tissues.

In the present study, we demonstrate that expression of Hif-2 cDNA under the control of an endothelial cell–specific promoter (Tie-2 promoter)²⁷ is sufficient to rescue vascular development in Hif-2^–/– embryos until embryonic day 14. 5 (E14.5). Thus, Hif-2 appears to be intrinsically required in endothelial cells. Furthermore, we show that Hif-2^–/– embryos have significantly reduced Tie-2 protein levels and that this defect can be rescued by the expression of Hif-2 cDNA under the control of the Tie-2 promoter. These observations raise the possibility that Tie-2 deficiency may be one of the underlying causes of the vascular defects in Hif-2a^–/– mice and that a recovery of Tie-2 expression induced by cDNA-encoded Hif-2 may be a contributing factor to the rescue of vascular development.

	Methods

Top Abstract Introduction Methods Results and Discussion References

 
Mice
The Animal Care Committee at the University of Connecticut Health Center approved all animal procedures. The procedure for tetraploid aggregation was essentially the same as described by Nagy et al.²⁸ Briefly, CD1 females (8 weeks, Harlan-Sprague Dawley, Indianapolis, Ind) were superovulated, and embryos were collected at the 2-cell stage. The 2 cells within each embryo were electrically fused in a CF-150 cell fusion apparatus (Biochemical Laboratory Services, Ltd) and incubated overnight in a cell culture incubator under 5% carbon dioxide to obtain 4-cell–stage tetraploid embryos. Every 2 tetraploid embryos were mixed with 8 to 12 embryonic stem (ES) cells in each microwell. After overnight incubation, the aggregates formed between ES cells and tetraploid embryos developed into blastocysts and were surgically transferred into the uteri of pseudopregnant females for further embryonic development.

DNA Manipulation
A plasmid that contained the Tie-2 promoter/enhancer was kindly provided by Dr Thomas N. Sato (University of Texas Southwestern Medical Center, Dallas, Tex). HIF-2 cDNA was a generous gift from Dr Stephen McKnight (University of Texas Southwestern Medical Center), and human alkaline phosphatase (hAP) cDNA was contributed by Dr Caiying Guo (University of Connecticut Health Center, Farmington, Conn). To construct the expression vector, the Hif-2 coding sequence was placed 5 to the internal ribosomal entry site (IRES)-hAP fragment to obtain an Hif-2-IRES-hAP cassette, which was inserted between the Tie-2 promoter and enhancer (Figure 1A). This construct will be referred to as Tie-2-Hif-2-hAP expression vector or Tie-2-Hif-2 transgene in the remainder of this report.

View larger version (89K): [in this window] [in a new window]   Figure 1. Tie-2 driven expression of Hif-2-hAP cassette. A, Schematic representation of Tie-2-Hif-2-hAP expression construct. After transcription, hAP serves as a faithful reporter because it is in same transcript as Hif-2. B to E, Phosphatase staining results: B, Mock-transfected ES cells; C, positive clone; D, Yolk sac membrane from embryo constructed from an ES cell line positive for reporter gene expression; E, Histological section of phosphatase-stained yolk sac. Arrowhead points to endothelial cells with positive staining signal. Bar dimensions: 100 µm in D; 20 µm in E.

To confirm the genotypes of embryos, genomic DNA was released from embryonic tissues and analyzed by polymerase chain reaction (PCR). Briefly, a small piece of embryonic tissue was dissected (avoiding posterior endoderm) and digested overnight at 55°C by proteinase K (50 mmol/L Tris-Cl, pH 8.0, 2 mmol/L EDTA, 1% Triton X-100, and 500 µg/mL proteinase K). After digestion, proteinase K was heat inactivated at 95°C for 5 minutes, and the digestion mixture was used directly for PCR at 1:25 dilution. The PCR strategy is schematically explained in Figure 2A. Cycling conditions for PCR reactions were as follows: 94°C for 30 seconds, 58°C for 45 seconds, and 72°C for 1 minute. Reactions were repeated for 35 cycles and analyzed on 1.2% agarose gels.

View larger version (39K): [in this window] [in a new window]   Figure 2. Confirmation of Hif-2–/– genotype of embryos derived from Hif-2–/– ES cells and Western blotting for Tie-2 and Flk-1. A, Schematic explanation of PCR strategy. To detect wild-type allele, 2 primers amplify a fragment in intron 2. This region and most of exon 2 are deleted in the targeted allele. To detect the mutant band, a pair of primers was used to amplify lacZ insert, which replaces deleted Hif-2 sequence in the targeted allele.22 B, Sample of PCR results. Lanes 1 to 6 are from embryonic tissues; lane 7 is from yolk sac membrane; lane 8 is from a wild-type embryo. C, Anti-Tie-2 and Flk-1 Western blots. Hif-2 genotypes in order of lanes 1 to 4 are as follows: +/+, +/–, –/–, and –/– with Tie-2 promoter–driven Hif-2 expression.

ES Cell Culture
ES cells were cultured essentially according to published procedures.²⁹ ES cell quality Dulbecco’s modified Eagle media (EmbryoMax; Specialty Media) was supplemented with sodium pyruvate (Invitrogen), nonessential amino acids (Invitrogen), leukemia inhibitory factor, antibiotics (penicillin and streptomycin; Invitrogen), ß-mercaptoethanol, and ES cell qualified 15% fetal bovine serum (Life Technologies, Inc, now part of Invitrogen). Hif-2^–/– ES cells were generated in a previous study²² and maintained on feeder cells (growth-arrested primary mouse embryonic fibroblasts). The Tie-2-Hif-2-hAP expression vector was introduced into Hif-2^–/– ES cells by transfection with Effectin reagent (Qiagen) according to the manufacturer’s instructions. Stably transfected ES cells were selected in ES cell media that contained 120 µg/mL hygromycin (Sigma-Aldrich). Approximately 400 hygromycin-resistant colonies were picked and expanded. To screen for ES cell lines expressing the reporter gene, ES cells were differentiated in vitro for 5 days according to a previously published protocol,³⁰ except that 48-well dishes were used to process several hundred clones in the same differentiation experiment. Differentiated ES cells were subject to alkaline phosphatase staining with a protocol described by Guo et al.³¹

Immunohistochemistry
Yolk sac membranes were fixed in 4% paraformaldehyde, rinsed several times with PBS, and dehydrated in increasing concentrations of methanol. The specimens were then treated with 5% hydrogen peroxide (in methanol), rinsed several times with PBS, and blocked in PBSMTN (PBS containing 2% dry milk, 1% Triton X-100, and 3% normal goat serum). Blocked specimens were incubated with rat antimouse CD31 antibody (Clone Mec13.3 at 5 µg/mL; BD Biosciences-Pharmingen) in PBSMTN, washed, and incubated with secondary antibody conjugated with horseradish peroxidase. Color development was performed in a solution that contained diaminobenzidine, hydrogen peroxide, and nickel chloride. For histological sectioning, the yolk sac membranes were dehydrated in increasing concentrations of ethanol, then toluene, and finally were embedded in paraffin. Sections were cut at 5 µm and counterstained with methyl green.

Western Blotting
Yolk sac tissues from E10.5 embryos were used for protein extraction, whereas embryonic tissues were used for genotype confirmation by PCR. To extract proteins, yolk sacs were homogenized in RIPA buffer (PBS containing 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, supplemented with complete protease inhibitors from Roche Applied Science). Proteins (50 µg/lane) were separated in 7.5% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. After blocking in TBSMT (20 mmol/L Tris-Cl, pH 7.5, 150 mmol/L NaCl, 2% dry milk, and 0.1% Tween-20), membranes were incubated with anti-Tie-2 (Pharmingen), anti-Flk-1, or anti-ß-actin (both from Santa Cruz Biotechnology) primary antibodies, washed in TBST (TBSMT minus milk), and developed by enzyme-linked chemiluminescence after incubation with secondary antibodies conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories, Inc). Band intensities were analyzed by the NIH image program.

Statistical Analysis
Proportions of surviving embryos were calculated for each population at specified developmental stages, and Hif-2^–/– populations were used as controls for comparison. Because the survival proportion cannot be less than zero, single-sided z tests were applied. Probability values <0.05 were accepted as statistically significant.

	Results and Discussion

Top Abstract Introduction Methods Results and Discussion References

 
Expression of Hif-2 Under the Control of the Tie-2 Promoter/Enhancer
To express Hif-2 cDNA in the endothelium, we placed the Hif-2 cDNA under the control of the endothelium-specific Tie-2 promoter/enhancer and introduced this construct into Hif-2^–/– ES cells by transfection (Figure 1A). We also fused, downstream of the Hif-2 cDNA, a cDNA fragment that encoded hAP so that transgene expression could be monitored by phosphatase staining. Both Hif-2 cDNA and the hAP sequence were under the control of the Tie-2 promoter and existed as 2 open reading frames in a bicistronic mRNA. Because hAP was the second open reading frame, it was preceded by an IRES for efficient translational initiation. This configuration ensured that the expression of the hAP reporter gene was tightly linked to that of the Hif-2 cDNA.

We screened for ES cell lines expressing the reporter gene by alkaline phosphatase staining after in vitro differentiation of transfected ES cells. We took this approach because in vitro differentiation of ES cells may generate endothelial cells, in which the Tie-2 promoter is active.³² Among 400 ES cell clones, 5 clones (1.25%) had significantly higher phosphatase activities than controls (Figures 1B and 1C).

Endothelium-specific expression of Tie-2–controlled transgene was demonstrated by phosphatase staining of embryos produced by aggregating tetraploid embryos with ES cell clones expressing the reporter gene (Figures 1D and 1E). Embryos obtained by this method are essentially ES cell–derived, because tetraploid cells cannot contribute to embryonic development with the exception of a very low percentage of cells in the posterior endoderm.^28,33 In extraembryonic tissues, tetraploid cells cannot contribute to mesoderm-derived cells, such as vascular endothelial cells. Conversely, ES cells cannot contribute to placental development, but they contribute to the embryo proper and mesoderm-derived tissues in the yolk sac. Such a complementarity dictates that the vascular system is ES cell–derived.

To confirm that the tetraploid aggregation procedure indeed gave rise to ES cell–derived embryos, we determined the genotypes of the embryos by PCR (Figures 2A and 2B). All DNA samples from the embryo proper resulted in PCR banding patterns that matched the known genotypes of ES cells, whereas the yolk sac DNA from –/– embryo also contained a faint wild-type band. The wild-type band presumably is derived from the endoderm, which is contributed by tetraploid cells.

Rescue of Hif-2^–/– Embryos by Endothelial Expression of Hif-2
In the previous study, Hif-2^–/– vascular defects were less variable in embryos derived directly from ES cells than in those obtained by breeding with ICR mice, presumably because ES cell–derived Hif-2^–/– embryos came from clonally pure ES cells and therefore had many fewer variations in genetic background. Consistency in the Hif-2^–/– phenotype was an important precondition to allow us to compare Hif-2^–/– embryos with and without Hif-2 cDNA expression. For this reason, we favored tetraploid/ES cell aggregation as the method to obtain embryos.

However, this method also has its limitations. The in utero survival of embryos generated by ES cell–tetraploid aggregation depends on technical factors in addition to the genotypes of the embryos. The primary technical limitation is that the totipotency of ES cells gradually decreases with increased number of ES cell passages. As a result, the developmental potential of ES cells is reduced with prolonged in vitro culturing.²⁸ Another major factor is electric pulse applied to 2-cell–stage embryos. A threshold level of electric potential and time duration is necessary to cause the fusion of 2 adjacent cells, but such conditions are also harmful to the cells. Because it is sometimes difficult to ascertain whether an individual embryo is dying owing to biological or technical reasons, we compared survival rates between different populations of embryos in addition to evaluating embryos on individual basis.

We chose to compare Hif-2^–/– embryos with or without the transgene at E12.5 on the basis of our previous finding that the vast majority of ES cell–derived Hif-2^–/– embryos developed vascular defects at E10.5 and died before E11.5. Although a very low percentage (10%) of embryos survived at E11.5, they all had severe vascular defects at this stage and died before E12.5.²² Thus, we assumed that if Hif-2^–/– embryos carrying the transgene were healthy at E12.5, they could be taken as evidence of successful rescue.

In the present study, we confirmed that among Hif-2^–/– embryos, even the least defective embryos had significant vascular abnormalities at E11.5 (Figures 3A and 3B). Although 10% of Hif-2a^–/– embryos had beating hearts, the majority of Hif-2a^–/– embryos were much smaller and necrotic by this stage. Furthermore, none of the 26 Hif-2a^–/– embryos survived to E12.5. The largest embryos had similar sizes to E11.5 embryos and were highly necrotic (Figures 3C and 3D). In contrast, 12 of 39 Hif-2^–/– embryos carrying the Tie-2-Hif-2-hAP expression vector were viable at E12.5 and displayed normal gross morphology (Figures 3E and 3F). In the Hif-2^+/– control, 11 of 27 embryos were viable at this stage. Thus, the survival frequency of the Hif-2^–/– embryos was raised to a level comparable to that of Hif-2^+/– embryos with the help of transgene expression.

View larger version (73K): [in this window] [in a new window]   Figure 3. Normal development of Hif-2–/– embryos rescued by Tie-2-Hif-2-hAP expression vector. A and B, Hif-2–/– embryos (without transgene) at E11.5. C and D, Hif-2–/– embryos (without transgene) at E12.5. E and F, Rescued embryos at E12.5 (yolk sac and embryo proper, respectively). G, Yolk sac covering body trunk of rescued embryo at E14.5. H, Anterior half of rescued embryo at E14.5. Bar dimensions: 1 mm in A, C, and E; 750 µm in B, D, and F; 2 mm in G and H.

We also examined whether Hif-2^–/– embryos could survive beyond E12.5 as a result of Hif-2 reexpression in the endothelium. From 51 deciduas examined at E14.5, we obtained 3 live embryos (Figures 3G and 3H). Without the transgene, all 57 deciduas contained severely decomposed tissues, which could not be dissected from deciduas without being broken into many tiny pieces. When Hif-2^+/– ES cells were used for the same assay, 5 of 31 deciduas contained live embryos. As shown in the Table, the rescue effect of Hif2a reexpression is statistically significant at both E12.5 and E14.5.

View this table: [in this window] [in a new window]   Statistical Analyses of Rescue Experiments

Although the proportion of surviving embryos was rather low at E14.5, such a low frequency may be explained by 2 factors discussed below. In mouse embryos, Hif-2 is also strongly expressed in the organ of Zuckerkandl, where it is essential for the regulation of catecholamine biosynthesis. In the C57/BL6 background, although Hif-2-(EPAS) deficiency does not cause severe vascular defects, Hif-2^–/– (EPAS^–/–) embryos still die at E13.5 because of reduced levels of catecholamine.²³ Thus, it is possible that some of the Hif-2^–/– embryos expressing the Tie-2-Hif-2 transgene might have died of a similar defect after E12.5. Although we could not obtain enough embryos after E12.5 to analyze the potential homeostasis defect, our previous investigations implied that catecholamine deficiency in Hif-2^–/– mice was not restricted to the C57/BL6 background.²²

Another plausible reason is that Hif-2^–/– ES cells carrying the transgene had gone through 3 rounds of single-colony selections: (1) during the original targeting experiment, (2) during the selection for –/– ES cells (+/– ES cells used in aggregation experiments were those that failed to become –/– during this selection, so that their in vitro culturing time was similar to –/– ES cells), and (3) transfection with Tie-2-Hif-2 transgene. Such prolonged in vitro culturing would undoubtedly reduce their developmental potential. Under such circumstances, it is remarkable that 3 embryos were viable at all at E14.5. Given the fact that none of the Hif-2^–/– embryos survived even at E12.5 even though –/– ES cells had a shorter in vitro culturing history than those carrying the transgene, and that the survival rate of Hif-2^+/– embryos was also significantly lower at E14.5 relative to earlier stages, we are inclined to conclude that endothelial expression of Hif-2 rescued Hif-2^–/– embryonic development until E14.5. The extent of recovery was limited by the quality of ES cells after prolonged in vitro culturing. The high passage number of these ES cells also prevented us from obtaining live-born mice to examine other interesting defects such as fetal respiratory distress syndrome due to insufficient surfactant production.²⁶ One caveat in the present experiments was that yolk sac endoderm was contributed by tetraploid cells that were wild-type for the Hif-2 gene. Nonetheless, it is highly unlikely that wild-type endoderm was responsible for the rescue activity. Hif-2^–/– embryos (without Hif-2 cDNA) were also produced by the same tetraploid aggregation procedure, but their yolk sac membranes were completely pale by E12.5 owing to severe hemorrhage (Figure 3C). In fact, crooked vascular patterns were already apparent at earlier stages (Figure 3A). Thus, we conclude that Tie-2–driven expression of Hif-2 in endothelial cells restored normal vascular development in mid–gestation-stage embryos.

To obtain further evidence that the development of the vascular system was indeed rescued, we examined the vascular system by immunohistochemical staining with anti-CD31(PECAM-1) antibody. Data shown in Figure 4 demonstrate normal vascular patterns in rescued Hif-2^–/– yolk sac tissues. In Hif-2^–/– embryos without the Tie-2-Hif-2-hAP expression vector, boundaries between individual vessels were fuzzy (Figure 4A). In histological sections, this is seen as excessive protrusion of endothelial cells into what would have been nonvascular tissues in normal situations (Figure 4B). The cellular basis for such an increased protrusive activity is not clear, but it may involve increased endothelial migration or abnormal endothelial/extracellular matrix interaction. In the yolk sac of rescued embryos, individual vessels are well separated from one another, and large vessels branch into progressively smaller sprouts (Figure 4C). Histological sections also demonstrate that vascular lumens were normal (Figure 4D).

View larger version (114K): [in this window] [in a new window]   Figure 4. Anti-CD31 staining of Hif-2–/– yolk sac membranes. A and B, Hif-2–/– (without rescue); C and D, Hif-2–/– (rescued). A and C are whole-mount stained specimens; B and D are sections. In A, boundaries between vessels are blurred, and development of vitelline vessels is compromised. B, Capillary-like vessels fuse extensively in Hif-2–/– yolk sac membranes (arrowhead), presumably because mutant endothelial cells protrude into nearby tissues. C, Normal vascular development after rescue. D, Discrete vascular lumens are formed (arrow). Bar dimensions: A and C, 400 µm; B and D, 50 µm.

We also investigated the mechanism of how Hif-2 regulates vascular development. Flk-1 and Tie-2 genes previously have been shown as downstream targets of Hif-2.^23,34 They are particularly relevant to the present study because their expression patterns are primarily endothelium specific,^35–37 and the rescue of Hif-2^–/– embryos was achieved through endothelium-specific expression of Hif-2. By Western blotting, we found that although Flk-1 was not significantly affected, Tie-2 was substantially reduced by Hif-2^–/– mutation (33±8% of Hif-2a^+/– embryos). More significantly, Tie-2 protein levels could be restored by endothelial reexpression of Hif-2 cDNA (69±13%; Figure 2C). These data raise the possibility that reduced Tie-2 expression may be responsible in part for the Hif-2 mutant phenotype and that the redemption of its expression may have helped rescued embryos to survive longer.

The Tie-2 promoter was not completely shut down by Hif-2^–/– mutation, because a reduced level of Tie-2 protein was still present. Thus, Tie-2-Hif-2 transgene expression could still be initiated. We speculate that once some Hif-2 protein was made, it might further upregulate the activity of Tie-2 promoter, and thus Tie-2-Hif-2 transgene expression was sustained in spite of a deficiency in endogenous Hif-2.

In summary, the present study demonstrates 2 important points. First, it illustrates the feasibility of confirming endothelium-specific functions of a widely expressed gene by lineage-specific reexpression in a null background. Second, the present data demonstrate an intrinsic role of HIF-2 in endothelial cells and raise the possibility that Tie-2 may be indeed a functionally relevant target for Hif-2. These findings further our understanding of how Hif-2 regulates vascular maturation and provide new opportunities to investigate the role of hypoxia in vascular development.

	Acknowledgments

 
This work was supported by National Institutes of Health grants P01 HL70694 and R01 HL68168. We are grateful to T.N. Sato for the Tie-2 promoter, S. McKnight for Hif-2 cDNA, and C. Guo for IRES/hAP cDNA cassette. We thank N. Ryan for technical assistance.

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