cell news 2/2013
10
werner risau prize
notch-dependent vegfr3 upregulation enables
angiogenesis without vegf–vegfr2 signalling
rui benedito
Blood and lymphatic vessels have been the subject of intense
investigation because they are seen as important therapeutic
targets in many types of cancer and in cardiovascular diseases
1,2
.
The formation of new blood vessels from pre-existing vessels,
a process named angiogenesis, requires the coordination of
several cellular and signalling mechanisms. The inner lining of
blood vessels is formed by endothelial cells that express several
surface receptors that are able to sense different extracellular
molecular cues. The vascular endothelial growth factor (VEGF)
family of secreted ligands and receptors are among the most
important and specifc regulators of endothelial cell sprouting,
proliferation and survival in a variety of organs and patholo-
gical processes
2,3
. In addition to being infuenced by external
factors, endothelial cells also have endogenous signalling me-
chanisms that can modulate their response to the surround-
ing environment. One such mechanism is the Notch signalling
pathway, where both ligands and receptors are transmembrane
proteins and upon cell-to-cell ligand-receptor activation, elicit
a transcriptional programme that leads to a change in the cell
status and behaviour of adjacent cells
4
.
Mutant mice lacking VEGF or Notch function in endothelial cells
die very early during development due to severe defects in the
genesis of the frst embryonic vessels
5-7
. VEGF is required for va-
scular sprouting and proliferation whereas activation of Notch
by the ligand Dll4 inhibits these processes
4,8
.
The effcient formation of new blood vessels requires properly
shaped angiogenic gradients that can activate only a few speci-
fc endothelial cells within an existing vascular network. These
frst responsive cells, take the leading position in a vascular sp-
rout and are known as endothelial tip cells. The other adjacent
endothelial cells follow the leader cell and are named by endo-
thelial stalk cells.
In the last years, several studies suggested that VEGF could in-
duce expression of the Notch ligand Dll4 in tip cells and ac-
tivation of Notch receptors by this ligand then repressed the
transcription of the most important VEGF receptor (VEGFR2/Kdr)
in adjacent stalk cells, reducing there angiogenic activity and
ability to outcompete tip cells in the cellular struggle to the
leading position. These results, led to the interpretation that this
negative feedback mechanism, of VEGF-to-Dll4 activation in
tip cells and Notch-to-VEGFR2 inhibition in stalk cells, is what
allows the selection of endothelial cells for sprouting and the
properly balanced heterogenous response of the endothelium to
the angiogenic stimuli
3,8
.
Most of the experiments supporting this strong retroregulation
between VEGF and Notch were performed with defned endo-
thelial cell lines (HUVECs)
in vitro
9-11
. However when we quanti-
fed the amplitude of regulation
in vivo
in animals with induced
deletion of
Vegfr2
in endothelial cells (
Vegfr2iDEC
), we observed
only a small decrease in Dll4 expression (Fig. 1a, b). We also
observed that when the Notch downstream transcriptional pro-
gram is impaired in endothelial cells, by deleting the transcrip-
tion factor
Rbpj
(
Rbpji
DEC
), there is no signifcant difference in
Vegfr2
transcription, protein levels or kinase activity when com-
pared with other genes expressed in endothelial cells and not
regulated by Notch, like Cdh5, or genes known to be regulated
by Notch in endothelial cells like Dll4 or Hey1 (Fig. 1b, c).
These results showed that endothelial cells with no VEGF/VEG-
FR2 signalling still express the Notch ligand Dll4 and hence still
have Notch signalling, therefore we decided to block Notch acti-
vity in vessels without VEGF signalling to see if that elicited any
endothelial response. According to the previous model, where it
was assumed that Notch controls the sensitivity of cells to VEGF,
we would predict that endothelial cells without VEGF signalling
cannot sprout or
proliferate, independently of the existing Notch
activity. However, we found that when we delete genes impor-
tant for Notch signalling in endothelial cells (
Dll4
or
Rbpj
), these
suddenly become much more resistant to the inhibition of VEGF
or deletion of
Vegfr2
(Fig. 1d, e). Our results show that normally
angiogenic endothelial cells are very sensitive to VEGF levels, but
in the absence of Notch they can still proliferate and sprout even
when VEGF signalling is impaired.
Apart from Vegfr2, previous studies showed that Notch can also
negatively regulate the transcription of another receptor from
