Chromogranin A (CGA)-derived polypeptide (CGA47-66) inhibits TNF-α-induced vascular endothelial hyper-permeability through SOC-related Ca2+ signaling

Abstract

CGA_1-78 (Vasostatin-1, VS-1) a N-terminal Chromogranin A (CGA)-derived peptide, has been shown to have a protective effect against TNF-α-induced impairment of endothelial cell integrity. However, the mechanisms of this effect have not yet been clarified. CGA_47-66 (Chromofungin, CHR) is an important bioactive fragment of CGA_1-78. The present study aims to explore the protective effects of CHR on the vascular endothelial cell barrier response to TNF-α and its related Ca²⁺ signaling mechanisms. EA.hy926 cells were used as a vascular endothelial culture model. The synthetic peptides CHR and CGA_4-16 were assessed for their ability to suppress TNF-α-induced EA.hy926 cells hyper-permeability through Transwell® and TEER assays. Changes in [Ca²⁺]_i were measured through confocal laser scanning microscopy. SOC channel currents (Isoc) were measured via patch-clamp analysis. RT-PCR and western blot were used to analyze mRNA and protein expression of the transient receptor potential channels TRPC1 and TRPC4, respectively. FITC and rhodamine-phalloidin fluorescence were used to assess cell morphology and the distribution of MyPT-1 and F-actin. Compared to untreated cells, TNF-α increased the permeability of EA.hy926 cells that was inhibited by pre-treatment with CHR (10-1000 nM) in concentration-dependent manner, and the effect was most obvious at 100 nM, but CGA_4-16 (100 nM) had no effect. TNF-α treatment increased the phosphorylation of MyPT-1 and stress fiber formation. CHR (10-1000 nM) pretreatment inhibited the cytoskeletal rearrangements and increased [Ca²⁺]_i in response to TNF-α treatment. CHR also reduced TRPC1 expression following TNF-α induction. Similar to SOC inhibitor 2-APB, CHR suppressed IP₃ mediated SOC activation. These findings suggest that CHR inhibits TNF-α-induced Ca²⁺ influx and protects the barrier function of vascular endothelial cells, and that these effects are related to the inhibition of SOC and Ca²⁺ signaling by CHR.

Keywords: Ca(2+); Chromofungin; Chromogranin A; SOC; TNF-α; Vascular endothelial cells.