Mechanical and Chemical Regulation of Arterial and Venous Specification - Mechanical and Chemical Signaling in Angiogenesis - page 9

3.3 Functional Interaction of Notch and VEGF Signaling
in Arterial Vessel Specification
As discussed in the previous sections, the VEGF-Notch axis is a central pathway in
specifying arterial fate. However, it had not been clear how VEGF signaling is
transduced into a Notch signal. This problem was addressed by a series of studies
using zebrafish models. As described in a Sect. 3.1 , grl is a zebrafish mutant where
the aorta is perturbed due to a mutation in a gene encoding a Notch signal target
transcription factor, Hey2. Thus, it is a suitable genetic mutation for studying
arterial specification. In a suppressor chemical screening using the grl mutant
zebrafish, two classes of small-molecule chemicals were identified [ 38 ]. One is
GS4012, a chemical that was found to interfere with extracellular signal-regulated
kinase (ERK). GS4012 was found to activate VEGFR by inhibiting ERK signaling.
The other class is PI3K/Akt inhibitors such as G4898, LY294002 and wartmannin.
GS4898 inhibits Akt, a downstream target of PI3K signaling. LY294002 and
wartmannin are chemical inhibitors of PI3K. These chemicals were able to rescue
grl phenotype by partial inhibition of PI3K which leads to activation of ERK [ 39 ].
Furthermore, it was shown that activated phosphorylated ERK is preferentially
expressed in arterial endothelial cells [ 39 ]. Inhibition of mitogen-activated or
extracellular signal-related protein kinase kinase (MEK), an upstream activator of
ERK, by either SL327 or U0126, results in loss of arterial endothelial cells and
improper aorta formation [ 39 ]. Inhibition of the VEGF signal pathway with a
VEGFR inhibitor 676475 leads to an overall reduction in the ERK activation and
defective arterial endothelial differentiation [ 39 ].
In another study, forward genetic screens have identified a zebrafish Plc-c
(phospholipase C-c) mutant that exhibits defects in the formation of arteries, but
not veins [ 40 ]. In this study, it was also shown that Plc-c mutant zebrafish embryos
failed to respond to exogenous VEGF expression. Injection of VEGF121 mRNA
into wild type zebrafish embryos resulted in ectopic expression of EphrinB2 in
veins, a Notch signal-dependent arterial marker. However, Plc-c mutant zebrafish
embryos injected with VEGF121 mRNA displayed either normal or reduced
EphrinB2 expression. In an independent cell culture study, it was reported that
PLC-c pathway activates ERK in the VEGF/VEGFR signal transduction cascade
[ 41 ]. These results indicate that VEGF/VEGFR induces arterial specification via
PLC-c/ERK signaling pathway which resides upstream of Notch signaling.
In contrast, the role of PI3K/Akt signaling in arterial fate determination requires
some caution. As discussed above, the arterial defect in grl mutant can be rescued
by the inhibition of PI3K signaling by small molecules [ 38 , 39 ]. Arterial specifi-
cation can be promoted by overexpression of dominant-negative Akt which results
in the inhibition of Akt, a downstream mediator of PI3K signaling [ 39 ]. Further-
more, constitutively active Akt induces venous endothelial fate [ 39 ]. These results
suggest that PI3K/Akt signaling inhibits arterial specification by interfering with
ERK activation, thus leading to venous cell fate. However, it is also reported that
PI3K signaling acts to induce Notch and Dll4 activation, the pathways for arterial