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Fatty acid carbon is essential for dNTP synthesis in endothelial cells

A Corrigendum to this article was published on 05 August 2015

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The metabolism of endothelial cells during vessel sprouting remains poorly studied. Here we report that endothelial loss of CPT1A, a rate-limiting enzyme of fatty acid oxidation (FAO), causes vascular sprouting defects due to impaired proliferation, not migration, of human and murine endothelial cells. Reduction of FAO in endothelial cells did not cause energy depletion or disturb redox homeostasis, but impaired de novo nucleotide synthesis for DNA replication. Isotope labelling studies in control endothelial cells showed that fatty acid carbons substantially replenished the Krebs cycle, and were incorporated into aspartate (a nucleotide precursor), uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates) and DNA. CPT1A silencing reduced these processes and depleted endothelial cell stores of aspartate and deoxyribonucleoside triphosphates. Acetate (metabolized to acetyl-CoA, thereby substituting for the depleted FAO-derived acetyl-CoA) or a nucleoside mix rescued the phenotype of CPT1A-silenced endothelial cells. Finally, CPT1 blockade inhibited pathological ocular angiogenesis in mice, suggesting a novel strategy for blocking angiogenesis.

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Figure 1: FAO stimulates vessel sprouting via EC proliferation.
Figure 2: CPT1A gene deletion in ECs causes vascular defects in vivo.
Figure 3: CPT1A silencing does not cause ATP depletion or redox imbalance.
Figure 4: CPT1A silencing reduces TCA replenishment and FAO is used for nucleotide synthesis.
Figure 5: Acetate or nucleosides rescue the CPT1AKD sprouting defect.
Figure 6: Most other cell types do not use fatty acid carbons for dNTP synthesis, and inhibition of CPT1A impairs angiogenesis.

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We thank M. Vander Heiden and D. Tollervey for discussion, R. Adams for providing VE-cadherin(PAC)-CreERT2 mice, and S. Rodríguez-Arístegui for synthesis of etomoxir. S.S. is funded by the Institution of Research/Innovation (IWT); R.M., B.G., A.R.C. and J.G. by the Research Foundation Flanders (FWO); U.B. by a Marie Curie-IEF Fellowship; K.C.S.Q. by CAPES (Brazil) and G.B. by KU Leuven. The work of S.-M.F. is supported by Marie Curie CIG, FWO-OdysseusII, Concern Foundation, Bayer Healthcare Pharmaceuticals. The work of P.C. is supported by IUAP7/03, Methusalem funding (Flemish Government), FWO grants, Foundation Leducq Transatlantic Network (ARTEMIS), Foundation against Cancer, European Research Council (ERC) Advanced Research Grant (EU-ERC269073) and AXA Research grant. S.Y.L. was supported by the Department of Defense CDMRP Visionary Postdoctoral Award (W81XWH-12-1-0466). Views and opinions of, and endorsements by, the authors do not reflect those of the US Army or the Department of Defense. The authors thank the MSU LC-MS Core.

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S.S., U.B., R.M., K.C.S.Q., G.B., I.E., A.Z., A.R.C., S.C., J.G., W.H., L.G., S.V., P.P.V.V., G.E., L.S., M.D., M.B., K.D.B., B.G., S.Y.L., S.-M.F. and P.C. contributed to the performance of the experiments and/or analysis of the data; H.G. provided advice; S.S., U.B., K.D.B., S.M.F. and P.C. designed the experiments; S.S., M.D., K.D.B., S.M.F. and P.C. wrote the paper; S.M.F. and P.C. conceptualized the metabolic analysis, and P.C. conceived and directed the study. All authors discussed the results and commented on the manuscript.

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Correspondence to Sarah-Maria Fendt or Peter Carmeliet.

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P.C. declares to be named as inventor on patent applications, claiming subject matter related to the results described in this paper.

Extended data figures and tables

Extended Data Figure 1 FAO regulates vessel sprouting.

a, mRNA expression of CPT1 isoforms (n = 3). b, CPT1A mRNA levels upon CPT1A silencing (CPT1AKD) (n = 11). c, Representative immunoblot of CPT1A for control and CPT1AKD ECs. d, FAO flux upon CPT1A silencing in venous (HUV) and arterial (HA) ECs, or upon small interference RNA transfection in venous ECs (siRNA) (n = 6 for HUV shRNA, n = 3 for HA shRNA and HUV siRNA). e, Schematic representation of FAO measurement using [9,10-3H]palmitate (reprinted from Immunity 35, Wang, R. et al. The transcription factor Myc controls metabolic reprogramming upon T lymphocyte activation, 871–882 (2011), with permission from Elsevier; ref. 24). f, Representative immunoblot for CPT1A upon genetic silencing of CPT1A using siRNA (siCPT1A). g, FAO flux upon silencing of CPT1C (CPT1CKD) (n = 3; P = NS). h, FAO flux levels in venous (HUV), arterial (HA) and microvascular (HMV) ECs (n = 4 for HUV versus HA and n = 3 for HUV versus HMV). i, Sprout number in control and CPT1AKD EC spheroids with mitomycin C (MitoC) treatment as indicated (n = 3). j, Flow cytometry counting of viable control and CPT1AKD ECs (n = 3). k, Analysis of random cell-motility tracks in control and CPT1AKD ECs (n = 4; P = NS). l, FAO flux and proliferation upon silencing of ACADVL (ACADVLKD) (n = 3 for each). m, Wound closure in control and ACADVLKD ECs (n = 3; P = NS). n, o, Quantification of vessel sprouting in control and ACADVLKD EC spheroids with MitoC treatment as indicated, total sprout length (n) and sprout numbers per spheroid (o) (n = 5). p, Sprout number in control and CPT1AOE EC spheroids with MitoC treatment as indicated (n = 3). q, Total sprout length in control and CPT1AOE EC spheroids treated with MitoC as indicated (n = 5). r, s, Representative phase (original magnification, 10× magnification) contrast images of control (r) and CPT1AOE (s) EC spheroids. t, Scratch wound assay in control and CPT1AOE ECs treated with MitoC as indicated (n = 3; P = NS). u, PCR analysis of genomic DNA from WT and CPT1AΔEC pups, confirming Cre-mediated recombination of the floxed Cpt1a allele as shown by the appearance of a 300-bp band. v, NG2+ area in neonatal vascular plexus of WT and CPT1AΔEC mice (3 litters, n = 8 pups for WT and 7 pups for CPT1AΔEC; P = NS). Data are mean ± s.e.m. of n independent experiments (a, b, d, gq, t) or the total number of mice (v). Statistical test: mixed models. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Extended Data Figure 2 CPT1A silencing does not cause cellular distress.

a, ADP/ATP ratio in control and CPT1AKD ECs (n = 3; P = NS). b, Sprout number upon oligomycin treatment (oligo) in control and CPT1AKD EC spheroids (n = 3). c, Glycolysis measurement in control and CPT1AKD ECs (n = 3; P = NS). d, Representative immunoblot for phosphorylated AMPK (pAMPK) and total AMPK (AMPK) and for LC3b I and II in control and CPT1AKD ECs. The ratio of the densitometrically quantified bands of pAMPK/AMPK and LC3b II/I is shown below the blots (n = 3; P = NS). e, Sprout number upon N-acetyl-cysteine (NAC) treatment in control and CPT1AKD EC spheroids (n = 3). f, g, Representative images (original magnification, 10×) of EC spheroids upon staining for TO-PRO3 in control (f) and CPT1AKD spheroids (g). h, i, Representative pictures (original magnification, 10×) of Hoechst/cleaved-caspase-3-stained control (h) and CPT1AKD (i) ECs. j, Representative immunoblots showing the ratio of phosphorylated (pATM)/total-ATM (ATM), p21/lamin and p53/lamin in control and CPT1AKD ECs. The ratios of the densitometrically quantified bands are shown below the blots (n = 3; P = NS). Data are mean ± s.e.m. of n independent experiments (ae, j). Statistical test: mixed models. NS, not significant. ****P < 0.0001.

Extended Data Figure 3 FAO is used for de novo nucleotide synthesis.

a, Schematic representation of the different carbon sources used for de novo synthesis of UMP. Note that palmitate contributes three carbons to the nine carbon skeleton of UMP. PRPP, 5-phosphoribosyl-1-pyrophosphate. b, Sprout number upon 5-fluorouracil (5FU) treatment in control and CPT1AKD EC spheroids (n = 4). c, Sprout number upon methotrexate (MTX) treatment in control and CPT1AKD EC spheroids (n = 4). d, Sprout number upon acetate treatment in control and CPT1AKD EC spheroids (n = 3). eg, Representative images (original magnification, 10×) of EC spheroids upon acetate treatment. h, i, Rescue of the sprouting defect of CPT1AKD spheroids by acetate was not affected by oligomycin treatment; panel h, total sprout length; panel i, sprout numbers/spheroid (n = 3; P = NS). j, k, Quantification of vessel sprouting using the EC spheroid model, showing that the reduction of total sprout length (j) and number of sprouts per spheroid (k) upon CPT1A silencing (CPT1AKD) was rescued by supplementation with a dNTP mix (n = 3). l, Quantification of MitoC-treated EC spheroid sprouting upon acetate or nucleoside mix supplementation (n = 3; P = NS). m, Glucose oxidation in ECs, measured by 14CO2 formation from [6-14C]glucose in control and CPT1AKD ECs (n = 4). n, Glutamine oxidation in ECs, measured by 14CO2 formation from [U-14C]glutamine in control and CPT1AKD ECs (n = 4; P = NS). o, Total contribution of [U-13C]glucose and [U-13C]glutamine to aspartate in control and CPT1AKD ECs (n = 3). p, [8-14C]hypoxanthine incorporation in RNA and DNA in control ECs (n = 3). Data are mean ± s.e.m. of n independent experiments (bd, hp). Statistical test: mixed models. NS, not significant. *P < 0.05, **P < 0.01, ****P < 0.0001.

Extended Data Figure 4 Etomoxir reduces vessel sprouting.

a, FAO flux upon etomoxir (Eto) treatment (n = 6). b, [3H]thymidine incorporation upon etomoxir treatment (n = 5). c, Scratch wound assay using MitoC-treated ECs upon etomoxir (Eto) treatment (n = 4; P = NS). d, Branch point quantification in the retinal vasculature of control (Ctrl) and etomoxir-treated (Eto) pups (8 litters, n = 24 pups for control and 16 for etomoxir treatment). e, f, Representative confocal images (original magnification, 10×) of retinal vessels stained for isolectin-B4 in control (e) and etomoxir (f) treated pups. g, Filopodia quantification in the retinal vascular front of control and etomoxir (Eto) treated pups (4 litters, n = 11 for WT and 9 for etomoxir; P = NS). Data are mean ± s.e.m. of n independent experiments (ad, g) or the total number of mice (d, g). Statistical test: mixed models. NS, not significant. ****P < 0.0001.

Extended Data Figure 5 Analysis of steady state.

Percentage M+2 or M+4 citrate and aspartate over different time points (24, 36, 48 and 52 h) after labelling with [U-13C]glucose (a), [U-13C]glutamine (b), or [U-13C]palmitate (c). Data are mean ± s.d. of n = 3 independent experiments.

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Schoors, S., Bruning, U., Missiaen, R. et al. Fatty acid carbon is essential for dNTP synthesis in endothelial cells. Nature 520, 192–197 (2015).

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