Cell News | Issue 02, 2017 - page 21

Cell News 01/2017 21
WERNER RISAU PRIZE 2017
olism. Since ECs rely on glycolysis for vessel branching1, we first
studied the effects of FOXO1 on this metabolic pathway. Trans-
duction of human umbilical vein endothelial cells (HUVECs) with
a FOXO1CA -encoding adenovirus (AdFOXO1CA) led to a robust re-
duction in glycolysis as evidenced by a reduction in extracellular
acidification rate (ECAR), glucose uptake, glycolytic flux and lac-
tate production (Fig. 3a–d). This metabolic phenotype correlates
with the reduced proliferation in FOXO1CA-expressing ECs and
raises a question as to whether FOXO1 promotes mitochondrial
oxidative phosphorylation. Surprisingly, FOXO1 did not stimulate
but instead diminished oxidative metabolism as indicated by a
decline in oxygen consumption in AdFOXO1CA-expressing HU-
VECs (Fig. 3e). Moreover, reactive oxygen species (ROS) forma-
tion were decreased (Fig. 3f). Importantly, FOXO1 did not induce
endothelial apoptosis, senescence, autophagy or energy distress
under the same experimental conditions (Fig. 3g). Together, our
data indicate that FOXO1 adapts metabolic activity to the lower
requirements of the quiescent endothelium. To gain insight into
the underlying mechanisms for this adaptability, we performed
transcriptome analysis of FOXO1CA- and GFP-transduced HU-
VECs. Gene set enrichment analysis (GSEA) revealed an enrich-
ment of the FOXO1 DNA-binding elements in genes induced by
FOXO1, while the MYC DNA-binding motif was highly enriched
in the repressed genes (Fig. 3h). Moreover, MYC target gene
signatures were downregulated in the FOXO1 transcriptome (Fig.
3i). Since MYC is a powerful driver of glycolysis, mitochondrial
metabolism and growth5, FOXO1 might antagonize endothelial
MYC signalling. In line with this, overexpression of FOXO1CA sup-
pressed MYC expression and protein levels in HUVECs (Fig. 3j–l).
Accordingly, numerous genes that are induced by MYC were
downregulated in FOXO1CA-overexpressing HUVECs, including
genes involved in cell metabolism and cell cycle progression (Fig.
3i, l). This regulation is in line with the repression of MYC by
FOXOs in cancer cells5-7 and points to MYC as a crucial effector
of FOXO1 in the coordination of endothelial metabolism and
growth. Remarkably, FOXO1 also induced the expression of neg-
ative regulators of MYC signalling including MXI1, an antagonist
of MYC transcriptional activity8, and FBXW7, an E3 ubiquitin
ligase that targets MYC for proteasomal degradation8 (Fig. 3l).
These data suggest that FOXO1 intersects with MYC signalling
at different levels. To explore further the role of MYC in ECs,
we analyzed the consequences of MYC inactivation for endo-
thelial metabolism. Bioenergetic analysis revealed that MYC
deficiency attenuated glycolysis and mitochondrial respiration
(Fig. 4a, b). Conditional deletion of Myc (Mycfl/fl)9 in mice using
the Pdgfb-creERT2 deleter impaired vascular expansion and led
to a thinned and poorly branched vasculature (Fig. 4c–e). These
phenotypes resemble the vascular defects in Foxo1iEC-CA mutant
mice and imply that MYC is a central component of endothelial
FOXO1 signalling. To test this directly, we attempted to rescue
the endothelial phenotypes imposed by FOXO1 activation by re-
storing MYC signalling with a Cre-inducible Myc overexpressor
allele (MycOE)10. Pdgfb-creERT2-induced overexpression of MYC
caused sustained vascular overgrowth and led to a profound in-
crease in EC number, proliferation and vessel density (Fig. 4f, g).
We then combined the MycOE, Foxo1CA and Pdgfb-creERT2 alleles
to generate endothelial-specific double mutants. Remarkably,
re-expression of MYC in ECs of Foxo1iEC-CA mice normalized the
hypobranched and hypocellular vascular phenotype caused by
FOXO1 activation (Fig. 4h, i). Moreover, coexpression of MYC
and FOXO1CA in HUVECs restored glycolysis and mitochondrial
respiration (Fig. 4j, k), indicating that regulation of MYC sig-
nalling by FOXO1 is critical for the coordination of endothelial
metabolism and growth.
This study identifies FOXO1 as a critical checkpoint of endothe-
lial growth that restricts vascular expansion. Our data suggest
that FOXO1 promotes endothelial quiescence by antagonizing
MYC, which leads to a coordinated reduction in the proliferative
and metabolic activity of ECs. The FOXO1-induced deceleration
of metabolic activity might not only enforce quiescence but also
support endothelial function. For instance, by lowering metabo-
lism, ECs will consume less energetic fuel for their homeostatic
needs, thereby ensuring efficient nutrient and oxygen delivery.
Reducing metabolic activity might also contribute to endothe-
lial redox balance. ECs are long-lived cells that need to protect
themselves against oxidative damage exerted by high oxygen
levels in the bloodstream. The FOXO1-induced reduction in
oxidative metabolism might thus be a mechanism to minimize
the production of mitochondria-derived ROS, thereby conferring
protection against the high-oxygen environment. Such a role
of FOXO1 in endothelial metabolism aligns with the broader
function of FOXOs in mediating oxidative stress resistance11-13,
and might also explain why ECs are exquisitely sensitive to a
change in FOXO1 status. It will be interesting to determine how
endothelial FOXO1 is regulated in vivo and how deregulation
contributes to disease.
About the author
Kerstin Wilhelm (PhD)
Max Planck Institute for Heart and Lung Research, Angiogen-
esis & Metabolism Laboratory, Ludwigstraße 43, 61231 Bad
Nauheim, Germany
E-mai:Kerstin.wilhelm@mpi-bn.mpg.de
10/2016 to Present: Postdoctoral fellow
Max-Planck Institute for Heart and Lung Research Bad Nauheim,
Germany
11/2012 - 09/2016 PhD Student
Max Planck Institute for Heart and Lung Research Bad Nauheim,
Germany
10/2010 - 10/2012 M.Sc. Molecular Medicine
Friedrich Schiller University, Jena, Germany
10/2007 - 09/2010 B.Sc. Biochemistry
University of Bayreuth, Bayreuth, Germany