159In a further work from our laboratory, we investigated this pathway also for CST
(Fornero et al. 2014 ). To this purpose, we studied CST colocalization with heparan
sulphate proteoglycans, the effect of CST on endocytotic vesicles trafficking and
caveolin 1 internalization, and the modulation of CST-dependent eNOS activation
on bovine aortic endothelial cells. Our results demonstrated that CST (5 nM) colo-
calizes with heparan sulphate proteoglycans and induces a marked increase in the
caveolae-dependent endocytosis and Cav 1 internalization; the effects of CST were
significantly reduced by pretreatment with heparinase or wortmannin. Our conclu-
sion was that, similarly to VS-1 (Ramella et al. 2010 ), the intracellular cascade acti-
vated by CST in endothelial cells depends on proteoglycans/PI3K-dependent
caveolae endocytosis acting as the initiating factor.
Moreover, CTS was unable to induce Ser^1179 eNOS phosphorylation after pre-
treatments with heparinase and methyl-β-cyclodextrin. These results are consistent
with the biochemical reports on CST, suggesting that this peptide, like other mem-
bers of the cell penetrating peptides (CPPs) family, exhibits membrane-interaction
properties because of both its amphipathic structure and extended hydrophobic
region. In particular, circular dichroism (CD) and nuclear magnetic resonance
(NMR) spectroscopy data indicated that CST folds into a short helical conformation
that interacts with membranes and causes considerable disordering at the level of
the phospholipid head groups. Moreover, two of the five residues of the helical
region of CST are arginines, an amino acid that has been proposed to form hydrogen
bond interactions with phospholipids (Sugawara et al. 2010 ).
Our experiments also show that CST activated endocytosis required the presence
of HSPGs on the surface of endothelial cells and that CST colocalizes with HSPGs.
The strong anionic charge present in proteoglycans makes them favorable binding
sites for cationic polymers, lipids, and polypeptides, which are used for drug and
gene delivery (Belting 2003 ; Rabenstein 2002 ). Negatively charged carbohydrates,
like HSPGs, located on the plasma membrane may serve as electrostatic traps for
the cationic CPPs (Jones and Howl 2012 ). Interestingly, the most prominent glycos-
aminoglycans on the surface of endothelial cells are precisely heparan sulphates and
one of the major protein core families of HSPGs is the membrane-bound glypicans,
that are enriched in caveolae, where a series of molecules involved with eNOS sig-
nalling are localized (Fleming 2010 ; Tarbell 2010 ). Furthermore, glypican-1 has
been hypothesized to be the mechanosensor for eNOS phosphorylation and activa-
tion in the shear stress induced response (Lopez-Quintero et al. 2009 ). It could be
speculated that the CST mediated membrane perturbation through HSPGs binding
and phospholipid interactions could resemble the acute membrane perturbation
involved in shear stress.
This matter, together with our previous finding of CST dependent eNOS activa-
tion (Bassino et al. 2011 ), led us to propose a CST induced mechanism of caveolae
endocytosis and consequent eNOS activation. These assumptions are supported by
our immunofluorescence experiments on Cav1 transfection and Cav1/eNOS and
colocalization. In particular, to test whether the endocytotic process triggered by
CST is caveolae-dependent, BAE-1 cells were transfected with GFP-Cav1 plasmid.
We observed that in transfected live cells GFP-Cav1 signal was confined in plasma
Signalling Pathways of CgA-Derived Peptides in Cardiac and endothelial cells