158
do not act directly on cardiomyocytes but, rather, on other cell types present in car-
diac tissue, presumably endothelial cells, as previously suggested by the experi-
ments performed on papillary muscles. Further experiments performed on Bovine
Aortic Endothelial cells (BAE-1) support the role of NO released by endocardial
endothelium in the effects exerted by VS-1 and CST. In particular, they indicated
that both VS-1 and CST promote the release of NO from endothelial cells by means
of a Ca2+-independent, PI3-K-dependent mechanism. Indeed, in contrast with ATP,
VS-1 and CST enhanced NO production with a mechanism that was independent of
intracellular Ca2+ concentration, and PI3-K blockade abolished the VS-1/CTS-
dependent NO increase in BAE-1 cells. Additional experiments performed on
BAE-1 cells reinforced the evidence of an endothelial NO production through a
Ca2+-independent, Akt-dependent eNOS phosphorylation (Bassino et al. 2011 ;
Gallo et al. 2007 ), a pathway previously reported in endothelial cells for insulin,
insulin-like growth factor-1, and oestrogens (Dimmeler et al. 1999 ; Hartell et al.
2005 ; Maniatis et al. 2006 ; Shaul 2002 ). Afterwards, we tested the hypothesis that
both peptides could induce a caveolae dependent endocytosis, resulting in Akt-
eNOS activation, by interacting with membrane heparan sulphate proteoglycans.
First we investigated this pathway for VS-1, grounding on its amphipathic proper-
ties and interactions with mammalian and microbial membranes (Kang and Yoo
1997 ; Maget-Dana et al. 2002 ). In fact, it has been shown that endocytosis plays a
major role in the signaling of different basic and amphipathic exogenous peptides,
that is, Antp, R9, and Tat (Duchardt et al. 2007 ). Our experiments performed on
BAE-1 cells clearly showed that VS-1 strongly increases endocytotic vesicles traf-
ficking, thus supporting the hypothesis that this peptide acts through a similar mech-
anism (Ramella et al. 2010 ). Moreover, given the critical requirement for the surface
Heparan Sulfate Proteoglycans (HSPGs) for endocytosis of cationic peptides (Poon
and Gariepy 2007 ), we supposed a receptorial-like role for HSPGs in the VS-1 path-
way. Along with this knowledge, we observed that HSPGs removal by treatment of
BAE-1 cells with heparinase completely abolished the VS-1-dependent endocyto-
sis. Moreover, heparinase also reverted the VS-1-induced displacement of caveolin
1 (Cav1) from plasma membrane to cytoplasm and the VS-1-dependent increase in
eNOS Ser^1179 phosphorylation. Since HSPGs and extracellular matrix also seem to
participate in the mechanosensing that mediates NO production in response to shear
stress (Florian et al. 2003 ), our results enhance the relevance of the HSPGs–NO axis
in the control of the vasomotor tone.
We also observed that VS-1-induced vesicles trafficking and Cav1 displacement
were both suppressed by Wortmannin, suggesting that the PI3K pathway plays a
central role in the VS-1-activated cellular signaling, by regulating the endocytotic
process, the Cav1 trafficking, and the eNOS phosphorylation mechanism. Our find-
ings are in agreement with the important role played by PI3K in membrane budding
and fission in endothelial cells (Li et al. 1995 ; Niles and Malik 1999 ). In addition,
both membrane remodeling and actin filament dynamics during endocytotic traffic
are strictly related to the PI3K/eNOS pathway, as suggested by the ability of guano-
sine triphosphatase dynamin to regulate vesicle scission and to interact with both
PI3K and eNOS, causing its activation (Schafer 2004 ).
G. Alloatti and M.P. Gallo