87a mechanism based on heparan sulfate proteoglycans interaction, caveolae endocy-
tosis and a PI3K-dependent eNOS phosphorylation has been proposed (Ramella
et al. 2010 ). Although the data so far generated provide some hints, further studies
are necessary to have a more complete picture on the mechanisms of the interaction
of CgA and CgA1-78 with endothelial cells.
5 The CgA-Dependent Angiogenic Switch
A growing body of evidence suggests that CgA and some of its fragments may have
a regulatory role in angiogenesis. For example, it has been shown that recombinant
full-length CgA can inhibit angiogenesis in the chick embryo chorioallantoic mem-
brane model (Crippa et al. 2013 ). Furthermore, CgA (0.1–0.2 nM) can inhibit capil-
lary sprouting induced by fibroblast growth factor (FGF)-2 or vascular endothelial
growth factor (VEGF), two potent pro-angiogenic factors, from rat aortic rings
(RAR) cultured in 3D-collagen gels (Crippa et al. 2013 ). Structure-function studies
showed that a functional site of CgA is located in the C-terminal region. However,
also CgA1-76, but not CgA1-373, 1–400 and 1–409, can inhibit VEGF- and FGF-
2- induced angiogenesis at 0.1–1 nM concentrations, suggesting that an additional
anti-angiogenic site is present in the N-terminal region, albeit in a latent (or less
active) form (Crippa et al. 2013 ). Interestingly, CgA1-78 was 30-fold less potent
than CgA1-76 (VS-I) in the RAR angiogenesis assay. It would appear, therefore,
that the anti-angiogenic site in the N-terminal region of CgA requires cleavage of
the first dibasic pair residues (K 77 K 78 ) and removal of C-terminal lysines for full
activation. Considering that biologically relevant levels of both CgA1-439 and VS-I
are present in circulation in normal subjects (see below), these findings suggest that
both molecules, but not large fragments of CgA lacking the C-terminal region, con-
tribute to the homeostatic inhibition of angiogenesis in normal conditions. The con-
cept that the N-terminal fragment of CgA is an anti-angiogenic molecule is also
supported by the observation that 0.3 μM CgA1-78 can inhibit in endothelial cells
hypoxia-driven morphological changes, vascular-endothelial (VE)-cadherin redis-
tribution, intercellular gap formation, tube morphogenesis, and hypoxia inducible
factor (HIF)-1α nuclear translocation (Veschini et al. 2011 ) as well as cell prolifera-
tion, migration and invasion induced by VEGF (Ferrero et al. 2004 ; Blois et al.
2006a; Di Comite et al. 2009a). Furthermore, a recent study has shown that CgA1-
78 can prevent choroidal neovascularization and vascular leakage in an established
mouse model of laser-induced ocular neovascularization (Maestroni et al. 2015 ),
suggesting that this polypeptide might have therapeutic activity in human ocular
diseases involving neovascularization or excessive vascular permeability.
Interestingly, while full-length CgA1-439 inhibit FGF-2−induced angiogenesis,
the fragments CgA1-373 and CgA352-372 (catestatin) can induce the release of
FGF-2 from endothelial cells and exert pro-angiogenic effects, pointing to CgA as
a paradoxical player in the regulation of angiogenesis. Indeed, CgA1-439, CgA1-
373 and CgA1-76 can counterbalance the pro-/anti-angiogenic activity of each
Chromogranin A in Endothelial Homeostasis and Angiogenesis