Vasostatin 1 activates eNOS in endothelial cells through a proteoglycan‐dependent mechanism

Abstract

Accumulating evidences point to a significant role for the chromogranin A (CgA)-derived peptide vasostatin 1 (VS-1) in the protective modulation of the cardiovascular activity, because of its ability to counteract the adrenergic signal. We have recently shown that VS-1 induces a PI3K-dependent-nitric oxide (NO) release by endothelial cells, contributing to explain the mechanism of its cardio-suppressive and vasodilator properties. However, the cellular processes upstream the eNOS activation exerted by this peptide are still unknown, as typical high-affinity receptors have not been identified. Here we hypothesize that in endothelial cells VS-1 acts, on the basis of its cationic and amphipathic properties, as a cell penetrating peptide, binding to heparan sulfate proteoglycans (HSPGs) and activating eNOS phosphorylation (Ser1179) through a PI3K-dependent, endocytosis-coupled mechanism. In bovine aortic endothelial cells (BAE-1 cells) endocytotic vesicles trafficking was quantified by confocal microscopy with a water-soluble membrane dye; caveolin 1 (Cav1) shift from plasma membrane was studied by immunofluorescence staining; VS-1-dependent eNOS phosphorylation was assessed by immunofluorescence and immunoblot analysis. Our experiments demonstrate that VS-1 induces a marked increase in the caveolae-dependent endocytosis, (115 +/- 23% endocytotic spots/cell/field in VS-1-treated cells with respect to control cells), that is significantly reduced by both heparinase III (HEP, 17 +/- 15% above control) and Wortmannin (Wm, 7 +/- 22% above control). Heparinase, Wortmannin, and methyl-beta-cyclodextrin (MbetaCD) abolish the VS-1-dependent eNOS phosphorylation (P(Ser1179)eNOS). These results suggest a novel signal transduction pathway for endogenous cationic and amphipathic peptides in endothelial cells: HSPGs interaction and caveolae endocytosis, coupled with a PI3K-dependent eNOS phosphorylation.

A new chromogranin A–dependent angiogenic switch activated by thrombin

“…9 Experimental evidence suggests that CgA1-78 can interact with heparan sulfate proteoglycans on endothelial cells and increase caveolae-dependent endocytosis. 36 CgA1-78 can also interact with membrane phospholipids, particularly with phosphatidylserine, an anionic phospholipid exposed on the surface of tumor endothelial cells, 10,[37][38][39] and inhibit VEGF-induced phosphorylation of ERK, an enzyme crucial for the induction of a cascade of events leading to increased cell proliferation and migration in angiogenesis. 40,41 In addition, CgA1-78 can inhibit TNF-induced phosphorylation of p38-MAPK by a pertussis toxin sensitive mechanism, a pathway important for the disassembly of adherence junctions in endothelial cells.…”

Phosphodiesterase type-2 and NO-dependentS-nitrosylation mediate the cardioinhibition of the antihypertensive catestatin

“…We showed that wortmannin blockade of the PI3K pathway abolished the CST-induced effects on inotropism and lusitropism. This suggests that, at least in part, the CST cardiotropism is achieved through this novel signaling pathway expected for receptor-orphan peptides with membrane-interacting properties, like CST (61).…”

Reduced serum levels of vasostatin-2, an anti-inflammatory peptide derived from chromogranin A, are associated with the presence and severity of coronary artery disease

“…Concentrations of vasostatin-1 and -2 in these experiments were chosen according to the previous literature. 30,31 Although higher than concentrations measured in vivo, the biological significance of such concentrations is in the possibility that tissue concentrations of these peptides are higher than plasma concentrations, and in the possibly lower biological activity of prokaryotic recombinant proteins here used (in this case from E. coli BL21) compared with eukaryotic proteins. In control cells, these proinflammatory factors induced a significant increase in VCAM-1, ICAM-1, and E-selectin expression ( Figure 5 and Supplementary material online, Figures S2 and S3).…”