Abstract
Endothelial cells (ECs) are plastic cells that can switch between growth states with different bioenergetic and biosynthetic requirements1. Although quiescent in most healthy tissues, ECs divide and migrate rapidly upon proangiogenic stimulation2,3. Adjusting endothelial metabolism to the growth state is central to normal vessel growth and function1,4, yet it is poorly understood at the molecular level. Here we report that the forkhead box O (FOXO) transcription factor FOXO1 is an essential regulator of vascular growth that couples metabolic and proliferative activities in ECs. Endothelial-restricted deletion of FOXO1 in mice induces a profound increase in EC proliferation that interferes with coordinated sprouting, thereby causing hyperplasia and vessel enlargement. Conversely, forced expression of FOXO1 restricts vascular expansion and leads to vessel thinning and hypobranching. We find that FOXO1 acts as a gatekeeper of endothelial quiescence, which decelerates metabolic activity by reducing glycolysis and mitochondrial respiration. Mechanistically, FOXO1 suppresses signalling by MYC (also known as c-MYC), a powerful driver of anabolic metabolism and growth5,6. MYC ablation impairs glycolysis, mitochondrial function and proliferation of ECs while its EC-specific overexpression fuels these processes. Moreover, restoration of MYC signalling in FOXO1-overexpressing endothelium normalizes metabolic activity and branching behaviour. Our findings identify FOXO1 as a critical rheostat of vascular expansion and define the FOXO1–MYC transcriptional network as a novel metabolic checkpoint during endothelial growth and proliferation.
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Data from microarray analysis have been deposited in the ArrayExpress repository under the accessions E-MTAB-4023 and E-MTAB-4025.
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Acknowledgements
We thank F. W. Alt and I. Aifantis for Mycfl/fl mice, and T. Enders, J. Sperling and K. Wilson for assistance with the mouse colony. The research of M.P. is supported by the Max Planck Society, the European Research Council (ERC) Starting Grant ANGIOMET (311546), the Deutsche Forschungsgemeinschaft (SFB 834), the Excellence Cluster Cardiopulmonary System (EXC 147/1), the Cluster of Excellence Macromolecular Complexes (EXC115), the Foundation Leducq Transatlantic Network (ARTEMIS), the LOEWE grant Ub-Net, and the European Molecular Biology Organization Young Investigator Programme. S.S. was supported by a PhD fellowship of the Belgian Science Policy (IWT) and I.M.A. is a recipient of a DOC-fFORTE fellowship of the Austrian Academy of Sciences. C.A.F was supported by a European Union FP7 Marie Curie Post-doctoral Fellowship and a FCT grant (IF/00412/2012). The research of K.R. is supported by an ERC Advanced Grant (268921) and C.K. received support from the National Institutes of Health K08CA090438. The research of H.G. is supported by Cancer Research UK, an ERC consolidator grant (REshape), the Foundation Leducq Transatlantic Network (ARTEMIS), the Lister Institute of Preventive Medicine and the British Council under the BIRAX Initiative. The work of P.C. is funded by long-term structural funding: Methusalem Funding by the Flemish Government, ERC Advanced Research Grant (269073), and FWO G.0834.13N from the Flanders Science Fund.
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K.W., K.H., G.E., S.S., M.F.O., R.L., B.Z., I.M.A., C.A.F., T.Bo. and M.P. performed experiments. K.W., K.H., G.E., S.S., M.F.O., R.L., B.Z., I.M.A., C.A.F., T.Bo., H.G., P.C. and M.P. analysed data. H.G., P.C. and M.P. guided research. T.Br., M.F., K.R., C.K., J.C.B. and H.G. provided essential reagents and protocols. K.H. contributed to manuscript writing and preparation. K.W., H.G., P.C. and M.P. wrote the paper. M.P. conceived and directed the study. All authors discussed the results and commented on the manuscript.
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Extended data figures and tables
Extended Data Figure 1 Constitutive and inducible deletion of Foxo1 in ECs of mice.
a, Strategy to generate a conditional Foxo1 mutant allele in which exons 2 and 3 are flanked by lox sites. The structures of the genomic locus, the targeting vector, and the targeted allele are shown. FRT-Neo-FRT, neomycin resistance cassette flanked by FRT sites. TK1, thymidine kinase. b, Table of viable offspring from Tie2-cre;Foxo1fl/+ (male) and Foxo1fl/fl (female) intercrosses. c, Control (Foxo1fl/fl) and Foxo1EC-KO mutants (Tie2-cre;Foxo1fl/fl) at E10.5. d, PCR of genomic DNA from P5 control (Foxo1fl/fl, lanes 1 and 3) and Foxo1iEC-KO (Pdgfb-creERT;Foxo1fl/fl, lanes 2 and 4) pups untreated (lanes 1 and 2) or treated (lanes 3 and 4) with 4-OHT. Recombination of the floxed Foxo1 allele (Δ) occurs only in 4-OHT-injected animals that are Pdgfb-creERT2-positive. e, Immunofluorescence staining for FOXO1, VE-cadherin (VECAD) and isolectin-B4 (IB4) in a P5 mouse retina of 4-OHT-injected control and Foxo1iEC-KO mice. f, Confocal images of mTmG+ control- and Foxo1iEC-KOmice that were injected with 4-OHT from P1 to P4 and analysed for GFP, ERG and IB4 expression.
Extended Data Figure 2 Endothelial FOXO1 deficiency leads to abnormal vessel size and shape.
a, Immunostaining for VECAD and ERG in Foxo1iEC-KO and control retinas. The bottom panels show the isolated VECAD and ERG signals of the inset. b, Confocal images showing maximum intensity projections and X–Y, X–Z, and Y–Z planes of a thick stack of IB4 and collagen IV (COL) stained P5 retinas. Foxo1iEC-KO mice develop enlarged vessels with abnormal lumen organization. White arrowheads point to areas with multiple vessel layers and intraluminal collagen strands. c, Images of IB4- (cyan) and TER119- (red) stained P5 retinas of control and Foxo1iEC-KO mice. Note that aggregates of TER119+ red blood cells form in Foxo1iEC-KO but not in control mice. d, Phospho-histone H3 (pHH3) and IB4 immunostaining of P5 Foxo1iEC-KO and control mice. e, Images of IB4-stained retinas at P21 showing an increased vessel density in Foxo1iEC-KO mice (same samples as in Fig. 1h). f, Higher magnification images of ERG-, ICAM2- and IB4-stained retinas at P21 showing increased numbers of ECs in the perivenous plexus of Foxo1iEC-KO mice. g, Bar graphs showing the mean endothelial area (n ≥ 8), mean diameter of central vein (n ≥ 8), and number of ERG/IB4+ cells (n ≥ 4) in P21 retinas of Foxo1iEC-KO and control mice. Data represent mean ± s.d. Two-tailed unpaired t-test. ****P < 0.0001.
Extended Data Figure 3 Inducible overexpression of a constitutively active FOXO1 mutant in ECs of mice.
a, A cassette containing the CAG promoter, a floxed STOP sequence, a cDNA encoding for Foxo1CA, and IRES-GFP was inserted into the Rosa26 locus. A schematic representation of the wild-type Rosa26 locus, the floxed allele, and the recombined allele after cre expression is shown. b, Immunofluorescence staining for FOXO1, GFP and PECAM in P5 Foxo1iEC-CA and control mice. c, Confocal images of mTmG+ control and Foxo1iEC-CA mice that were injected with 4-OHT from P1 to P4 and analysed for GFP, ERG and IB4 expression. The right half of both images shows the GFP signal alone. d, High-magnification images of IB4-stained retinal vessels at the angiogenic front in control and Foxo1iEC-CA pups. e, BrdU and IB4 labelling of whole-mount P5 retinas reveals reduced endothelial proliferation in Foxo1iEC-CA animals. f, Confocal images showing MYC and PECAM immunostaining in P5 retinas of control and Foxo1iEC-CA mice. The lower half of both images shows the MYC signal alone. g, Quantification of FOXO1 nuclear staining intensity in ECs (n = 3), radial migration (n = 10), endothelial coverage (n = 10), branch points (n = 10), and endothelial BrdU incorporation (n ≥ 6) in P5 retinas of control and Foxo1iEC-CA mutant mice. Data represent mean ± s.d. Two-tailed unpaired t-test. **P < 0.01; ***P < 0.001; ****P < 0.0001.
Extended Data Figure 4 FOXO1 restricts EC propagation and vascular growth in a cell-autonomous manner.
a, Timeline for the analysis of angiogenesis in the embryonic hindbrain. Plug-positive female mice were injected with 4-OHT from E8.5 to 10.5 and embryos harvested on E11.5 for hindbrain dissection. b, Confocal images of E11.5 control and Foxo1iEC-CA hindbrains stained with IB4 and GFP. c, High-magnification images of IB4-stained blood vessels in the ventricular zone of control and Foxo1iEC-CA mice. d, Timeline for the analysis of control and Foxo1iEC-CA low-degree chimaeras that heterozygously co-express the mTmG Cre reporter. Control and Foxo1iEC-KO mice were injected with a single low dose of 4-OHT at P3 and retinas analysed at P5. e, f, Confocal images (e) and quantification (f) of control;mTmG and Foxo1iEC-CA;mTmG retinas after low-dose 4-OHT treatment at P3 (n = 9). Samples were labelled for GFP, ERG and IB4. Data represent mean ± s.d., two-tailed unpaired t-test. ****P < 0.0001.
Extended Data Figure 5 Forced expression of FOXO1 does not induce apoptosis, senescence, autophagy or energy distress in cultured ECs.
a, Immunoblot analysis and quantification of FOXO1 protein levels in AdCTL and AdFOXO1CA–Flag transduced HUVECs (n = 20). b, ATP levels in ECs 24 h after transduction with AdCTL or AdFOXO1CA (n = 7). c, Western blot images and quantification of AdCTL- or AdFOXO1CA–Flag-transduced HUVECs showing that FOXO1CA does not alter the phosphorylation of AMPKα (Thr 172) or of its substrate ACC (Ser 79). Oligomycin (Oligo), positive control. TUB, tubulin. n = 10. d, Western blotting of AdCTL- or AdFOXO1CA–Flag-transduced HUVECs illustrating that overexpression of FOXO1CA does not induce apoptotic cell death. Cleaved caspase3 (CASP3) and PARP served as markers of apoptosis. Cycloheximide (CHX) and TNF-α (TNF) costimulation, positive control. e, Analysis of senescence-associated genes by microarray demonstrating that senescence markers were not significantly changed or even downregulated in FOXO1CA-overexpressing ECs. n = 3. f, Images of β-galactosidase stainings in AdCTL- and AdFOXO1CA-transduced HUVECs showing no increase in senescence-associated β-galactosidase activity (SABG). g, Densitometric quantification of the LC3-II to LC3-I ratio in AdCTL- or AdFOXO1CA-transduced HUVECs (n = 10). h, Immunofluorescence analysis of AdCTL- and AdFOXO1CA-transduced HUVECs (both coexpressing GFP) using LC3 and GFP antibodies. Chloroquine (CQ), positive control. DAPI, endothelial nuclei. Data in a–c, e and g represent mean ± s.d. Two-tailed unpaired t-test. *P < 0.05; ****P < 0.0001; NS, not significant.
Extended Data Figure 6 FOXO1 represses MYC signalling in ECs.
a, Microarray expression analysis of FOXO1 and of canonical FOXO target genes in AdFOXO1CA- and AdCTL-expressing HUVECs 16 h after transduction (n = 3). b, GSEA of the FOXO1 DNA-binding element (TTGTTTAC) gene set in AdFOXO1CA- or AdCTL-transduced ECs. ES, enrichment score; NES, normalized enrichment score. c, GSEA of MYC gene signatures39,40,41,42 showing the downregulation of MYC target genes in FOXO1CA-expressing HUVECs. d, qPCR expression analysis of MYC at 3, 6 and 16 h in AdCTL and AdFOXO1CA-transduced HUVECs (n ≥ 4). e, qPCR analysis (n = 4) of Myc mRNA levels in ECs isolated from Foxo1CA mice 24 h after transduction with a control or Cre (AdCre) adenovirus. f, Immunoblot analysis of MYC in ECs isolated from Foxo1CA mice following transduction with AdCTL or AdCre (n = 3). Cre-mediated recombination gave rise to a 2.8 ± 0.3-fold increase in FOXO1 protein expression. g, Expression analysis of MYC in HUVECs by western blotting after RNA interference (RNAi)-mediated knockdown of FOXO1 (siFOXO1). siSCR, scrambled control (n = 3). h, MYC protein expression in ECs isolated from Foxo1fl/fl mice 24 h after transduction with an AdCTL or AdCre-encoding adenovirus (n = 3). a, d–h, Data represent mean ± s.d., two-tailed unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Extended Data Figure 7 FOXO1 interferes with MYC signalling at different levels.
a, Western blot analysis of MYC, MXI1 and FBXW7 in AdCTL- or AdFOXO1CA–Flag-transduced HUVECs. b, Immunoblot analysis and quantification of MYC protein levels in AdCTL and AdFOXO1CA–Flag transduced HUVECs that were co-treated with the proteasomal inhibitor MG132 (n = 3). c, Analysis of MYC protein half-life in AdCTL- or AdFOXO1CA–Flag-transduced HUVECs. The day after transduction, HUVECs were treated with cylcoheximide (CHX) and incubated for the times indicated. Data represent mean ± s.d. Two-way ANOVA with Bonferroni’s multiple comparison post-hoc test. d, e, qPCR (d) and immunoblot analysis (e) of MYC levels in control (siSCR) or MXI1 (siMXI1) siRNA-transfected HUVECs that were also transduced with AdCTL or AdFOXO1CA–Flag (n ≥ 5). f, qPCR analysis of MYC target genes in siSCR or siMXI1-transfected HUVECs that were cotransduced with AdCTL or AdFOXO1CA (n ≥ 3). Data represent mean ± s.d. One-way ANOVA with Bonferroni’s multiple comparison post-hoc test was performed in b, d, e and f. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Extended Data Figure 8 MYC regulates genes involved in cell metabolism and growth in ECs.
a, b, Analysis of MYC expression by qPCR (a) and immunoblot (b) in scrambled (siSCR) and MYC (siMYC) siRNA-treated HUVECs 24 h after transfection (n = 7). c, GSEA of the MYC (CACGTG) DNA-binding element gene set in siSCR- or siMYC-transfected HUVECs. d, GSEA of MYC gene signatures39,40,41,42 showing the downregulation of MYC target genes in MYC-depleted HUVECs. e, Heat map of downregulated MYC signature genes in MYC-silenced HUVECs (n = 3). Genes highlighted in red indicate genes that are also suppressed by FOXO1CA overexpression. f, Table of KEGG gene sets enriched among genes downregulated in the MYC siRNA-transfected ECs. g, Expression analysis of FOXO1-regulated MYC target genes by qPCR in MYC-silenced HUVECs (n ≥ 4). a, g, Data represent mean ± s.d., two-tailed unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Extended Data Figure 9 MYC is a critical driver of endothelial proliferation, growth and metabolism.
a, b, IB4 and pHH3 (a) or BrdU (b) labelling of P5 retinas reveals reduced endothelial proliferation in MyciEC-KO mice. c, ICAM2, IB4 and COL staining of retinas at P5 showing an increased number of empty (COL+, IB4−) sleeves (white arrows) in the plexus of MyciEC-KO mutants. d, Quantitative analysis of the indicated vascular parameters in P5 retinas of control and MyciEC-KO mice (n ≥ 8). e, ECAR (n = 4) and OCR (n = 4) in AdMYC-transduced HUVECs showing a heightened metabolic activity in MYC-overexpressing ECs (6.8 ± 1.4-fold MYC overexpression). f, Pdgfb-creERT2-mediated overexpression of MYC (2.4 ± 0.8-fold MYC overexpression) enhances vascular growth as indicated by the parameters assessed at P5 (n ≥ 6). g, ERG and IB4 labelling of P5 retinas showing an increase in cellularity in vessels of MyciEC-OE mice. h, Enhanced EC proliferation in MyciEC-OE mice as revealed by BrdU and IB4 costaining. i, j, Overview (i) and higher magnification images (j) of ICAM2-, IB4- and COL-stained retinas at P21 showing aberrant vascular growth and venous enlargement in MyciEC-OE mice. k, Increased endothelial cellularity in veins of MyciEC-OE mice at P21. d–f, Data represent mean ± s.d., two-tailed unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Extended Data Figure 10 Restoration of MYC signalling in FOXO1CA-overexpressing endothelium normalizes vascular growth.
a, b, Confocal images (a) and quantification (b) of ERG- and IB4-stained P5 retinas in control, Foxo1iEC-CA, MyciEC-OE and Foxo1iEC-CA/MyciEC-OE mice (same samples as in Fig. 4h) showing that EC numbers are normalized in the Foxo1iEC-CA/MyciEC-OE double mutants (n ≥ 3). c, Relative ROS levels in AdCTL-, AdFOXO1CA-, AdFOXO1CA/AdMYC- and AdMYC-transduced HUVECs showing that ROS levels increase again in FOXO1CA/MYC co-expressing ECs (n ≥ 6). b, c, Data represent mean ± s.d., one-way ANOVA with Bonferroni’s multiple comparison post-hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
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Wilhelm, K., Happel, K., Eelen, G. et al. FOXO1 couples metabolic activity and growth state in the vascular endothelium. Nature 529, 216–220 (2016). https://doi.org/10.1038/nature16498
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DOI: https://doi.org/10.1038/nature16498
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