MetaboLights

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acid d2701_GlycolysisHexose_6-phosphatePyruvic_acid02_Krebs CycleFumaric_acidCitric_acidL-Malic_acidOxoglutaric_acidSuccinic_acidOxalacetic_acid03_Amino AcidsL-Glutamic_acidL-GlutamineL-MethionineGlycineL-AlanineL-ProlineL-ValineIle_LeuL-AsparagineL-CysteineLAspartic_acid05_NucleotidesADPAMPATPCDPCMPCTPGDPGMPGTPTDPTMPTTPUDPUMPUTP07_RedoxGlutathioneNADNADHNADPNADPHOxidized_glutathione10_CoAAcetyl-CoAXX_SpecialUreidosuccinic acidAICARInosinic acidL-Dihydroorotic acidOrotic acidSAICARXanthylic acidMetaboLightsPublicLiquid Chromatography MS - negative - hilicMeasurements were performed using a Dionex UltiMate 3000 LC System (Thermo Fisher Scientific) in-line connected to a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) (Huang et al., 2017). 40 μl of sample was injected and loaded onto a SeQuant ZIC/pHILIC Polymeric column (Merck- Millipore). A linear gradient was carried out starting with 84% solvent A (95-5 acetonitrile- H2O, 2 mM ammonium acetate pH 9.3) and 16% solvent B (2 mM ammonium acetate pH 9.3). From 2 to 29 min, the gradient changed to 75% B and was kept at 75% until 34 min. Next, a decrease to 16% B was carried out to 42 min, and then, 16% B was maintained until 58 min. The solvent was used at a flow rate of 100 μl/min, and the column temperature was kept constant at 25°C.Endothelial SLC25A13 links glutamine metabolism to angiogenesis.Patricia Yague SanzLaboratory of Angiogenesis and Vascular Metabolism, Center for Cancer Biology (CCB), VIBHUVEC cellmass spectrometry assayMetabolites from 3 × 105 cells were extracted in 300 μL cellular extraction buffer (80% methanol with 2 μM d27 myristic acid). Next, the extracts were centrifuged at 4°C for 15 min at 20,000 × g and the supernatants were used for Liquid chromatography–mass spectrometry (LC-MS) (ion-pairing mode).Homo sapienshttps://www.ebi.ac.uk/metabolights/MTBLS15306Joris Souffreau.Mieke Dewerchin.Els Gils.Anh-Co Truong.Patricia Yague Sanz. VIB-KU Leuven Center for Cancer Biology. patriciayaguesanz@gmail.com.Abhishek Subramanian.Koen Veys.Ling Jin.Peter Carmeliet. VIB-KU Leuven Center for Cancer Biology. peter.carmeliet@kuleuven.be.Mathias Declercq.Huberte Moreau.Anne Cuypers.Baharak Hosseinkhani.Massimiliano Mazzone.Sabine Wyns.Bart Ghesquière.Liliana Sokol.Mila Borri.Stefan Vinckier.Guy Eelen.Mira Mousa.Pieter-Jan Coolen.Katleen Brepoels.Data collection (total metabolite abundances as well as 13C isotopic incorporation) was performed with the Xcalibur software (Thermo Fisher Scientific). For calculation of the total carbon/nitrogen contribution, we corrected for naturally occurring isotopes using the method of Fernandez et al. (Fernandez et al., 1996). For relative metabolite levels, the total ion count was normalized to the protein content. TreatmentSample preparationDonorReplicateGenotypepatriciayaguesanz@gmail.comFor 13C-carbon incorporation from glutamate in metabolites, HUVECs were incubated for 48 hours with [U-13C] glutamine in a 100% labeling way (glucose: 5.5 mM; glutamine: 2 mM). Metabolites from 3 × 105 cells were extracted in 300 μL cellular extraction buffer (80% methanol with 2 μM d27 myristic acid). Next, the extracts were centrifuged at 4°C for 15 min at 20,000 × g and the supernatants were used for Liquid chromatography–mass spectrometry (LC-MS) (ion-pairing mode). The medium for all conditions was harvested before extraction.MetabolomicsMetabolomicsHUVEC cellThermo Scientific Q Exactivetargeted analysisThermo Scientific Dionex UltiMate LC systemangiogenesisHomo sapiensexperimental blankmetabolic processVIB-KU Leuven Center for Cancer Biologyexperimental sampleSLC25A13MetabolomicsHUVEC cellThermo Scientific Q Exactivetargeted analysisThermo Scientific Dionex UltiMate LC systemangiogenesisHomo sapiensexperimental blankmetabolic processVIB-KU Leuven Center for Cancer Biologyexperimental sampleSLC25A13The mass spectrometer operated in negative ion mode; settings of the HESI probe were as follows: sheath gas flow rate at 25, auxiliary gas flow rate at 5 (at a temperature of 260°C). Spray voltage was set at 4.8 kV, temperature of the capillary at 300°C, and S-lens RF level at 50. Normal full scan (resolution of 35,000 and scan range of m/z 50–1,050) was applied. In case of low metabolite levels (such as dNTPs), a targeted Selected Ion Monitoring (SIM) method was applied.falseEndothelial SLC25A13 links glutamine metabolism to angiogenesisAttention to exploring endothelial cell (EC) metabolism is increasing, but a holistic perspective on the metabolic adaptations of ECs during angiogenic proliferation, is still missing. Here, we use a previously in-house developed computational EC-adapted genome scale metabolic model that recapitulates known metabolic features in proliferating ECs to predict novel pathways central to biomass synthesis. We unveil the metabolic function of the glutamate-aspartate antiporter SLC25A13, previously overlooked in ECs, as a key regulator of glutamine utilization and provide evidence regarding its contribution to the generation of cholesterol and other biomass precursors. To our knowledge, predominant SLC25A13-linked utilization of glutamine for cholesterol synthesis has not been reported in other cell types in prior literature. Silencing of SLC25A13 in ECs reduced proliferation and vessel sprouting in vitro as well as in a choroidal neovascularization mouse model. These insights underscore the particular characteristics of EC metabolism and may warrant further evaluation of SLC25A13 as a putative new anti-angiogenic target.2026-08-102026-08-10MTBLS15306