Cell News 02/2017
20
Endothelial cells (ECs) line the luminal side of blood vessels.
When tissues are in need of oxygen and nutrients they secrete
proangiogenic factors, which trigger ECs to become invasive
and protrude filopodia. The so-called tip cells lead the sprouts
and extend their filopodia towards the source of the angiogenic
signal. Tip cells are followed by stalk cells, which proliferate to
elongate the sprout. Eventually, tip cells connect with tip cells
from adjacent sprouts to establish new vessel circuits. This
process of vessel growth from pre-existing vessels is named
angiogenesis. Angiogenesis continues until nutrient and oxygen
supply meets tissue demand, proangiogenic molecules are
silenced, and ECs become quiescent again. Compared to quies-
cence active sprouting is highly energy demanding
1
. While the
tip cells need an extensive amount of ATP, because they have to
remodel their cytoskeleton in order to protrude filopodia, stalk
cells need to double their biomass in order to be able to divide
and proliferate. This suggests that ECs have to adapt their me-
tabolism when switching from quiescence to vascular growth.
However, how ECs couple their metabolic activity to growth
state is poorly understood at the molecular level.
We analyzed the role of FOXO in the endothelium. FOXO is an
effector of the phosphatidylinositol-3-OH kinase (PI(3)K)/AKT
pathway that links growth and metabolism. PI(3)K signalling
inhibits FOXOs through AKT-mediated phosphorylation leading
to their nuclear exclusion. We investigated the role of FOXO1 in
ECs, an enriched FOXO family member in the endothelium. To
this end, we bred floxed
Foxo1
mice (
Foxo
1
fl/fl
)
2
with a
Tie2-cre
deleter, which recombines in endothelial and haematopoietic
cells.
Tie2-cre
-mediated deletion of
Foxo1
(
Foxo1
EC-KO
) caused
defective vascular development and embryonic lethality around
embryonic day (E)10.5
3
, suggesting that endothelial FOXO1 is
essential for embryo development. Immunofluorescence analysis
of developing blood vessels in the postnatal retina showed high
levels of FOXO1 expression in the endothelium (Fig. 1a). Further
examination of the subcellular distribution revealed a diffuse
nucleocytoplasmic localization of FOXO1 at the angiogenic
front, where most of the EC proliferation occurs, but a stron-
ger nuclear pattern in the plexus, where vessels remodel, and
endothelial proliferation abates (Fig. 1a). This spatial difference
in subcellular localization suggests that FOXO1 is important
for governing endothelial growth. To test this, we assessed
the impact of
Foxo1
deletion on retinal angiogenesis using
the tamoxifen-inducible, endothelial-selective
Pdgfb-creERT2
line (
Foxo1
iEC-KO
). Endothelial loss of
Foxo1
caused a dense and
hyperplastic vasculature and resulted in the inability of ECs to
extend proper sprouts (Fig. 1b–f). Instead, ECs grew in clusters
leading to vessel enlargement and blunting of the angiogenic
front (Fig. 1d, f). Strikingly, numerous filopodial bursts were
emanating from the stunted front (Fig. 1c, d), suggesting that
FOXO1 deficiency results in uncoordinated vascular growth. As-
sessment of 5-bromodeoxyuridine (BrdU) incorporation demon-
strated a substantial increase in endothelial proliferation in the
Foxo1
iEC-KO
mutants (Fig. 1g, j), indicating that deregulated pro-
liferation drives this aberrant vessel phenotype. Because of the
fact that in the majority of mouse mutants the retinal vascular
phenotypes resolve during later stages of retinal vascular devel-
opment, we analyzed the
Foxo1
iEC-KO
mutants at postnatal day 21
(P21) when overall vessel morphogenesis is completed. Impor-
tantly, the vascular defects did not normalize at later stages of
development, but showed a persistent increase in endothelial
number, density and vessel diameter (Fig. 1h, i). We conclude
that FOXO1 is a suppressor of endothelial growth and prolifera-
tion, whose inactivation leads to uncontrolled overgrowth.
Next, we determined the consequences of FOXO1 activation
in ECs. We used a Cre-inducible gain-of-function allele (
Foxo-
1
CA
) in which the AKT phosphorylation sites are mutated, thus
rendering FOXO1 constitutively nuclear (Fig. 2b)
4
.
Tie2-cre
-me-
diated expression of this IRES-GFP-coexpressing mutant (
Fox-
o1
EC-CA
) was incompatible with embryo survival beyond E10.5
(Fig. 2a), highlighting the sensitivity of ECs towards changes
in FOXO1 status. We then used the Pdgfb-creERT2 strain to
express
Foxo1
CA
in the retinal endothelium (
Foxo1
iEC-CA
). Immu-
nofluorescence studies revealed an enriched FOXO1 signal in
endothelial nuclei and confirmed the EC-specific expression of
GFP (Fig. 2b). Forced activation of FOXO1 led to a sparse and
hyperpruned vascular network that contained fewer ECs (Fig.
2c, d, f–h). These retinal vessels established a lumen but were
thinner (Fig. 2g, h). Staining for phospho-histone H3 (pHH3)
revealed a reduction in EC proliferation in
Foxo1
iEC-CA
mice while
endothelial apoptosis was not altered (Fig. 2e, f, i). Given the
fact that different vascular beds might cope variably with forced
FOXO1 expression, we investigated the consequence of FOXO1
activation in the embryonic hindbrain. Similar phenotypes were
observed in the hindbrain vasculature (Fig. 2j, k), indicating that
FOXO1 is a critical driver of endothelial quiescence.
We next assessed whether FOXO1 regulates endothelial metab-
WERNER RISAU PRIZE 2017
Kerstin Wilhelm
FOXO1 couples metabolic activity and growth state
in the vascular endothelium
Max Planck Institute for Heart and Lung Research,
Angiogenesis & Metabolism Laboratory, Bad Nauheim, Germany
