Chr Inhibits TNF-A-Induced Increase In Vascular Endothelial Hyperpermeability By Calcium Signal Pathway Mechanism

Objective:To investigate the influence of Chromogranin A (CGA) derived-peptide chromofungin (CHR) on vascular endothelial cells hyperpermeability induced by tumor necrosis factor alpha (TNF-a) through calcium signal pathway mechanism.Methods:The human umbilical vein endothelial cell line (EA.hy926) was employed as the research object, CHR and TNF-awere used as stimulus.1) The monolayer permeability of EA.hy926 cells was measured by detecting absorbance (A value) of HRP in the lower compartment of Transwell; 2) The expression and distribution of F-actin in EA.hy926 cells were observed by cellular immune fluorescence and laser scanning confocal microscopy; 3) The intracellular Ca concentration ([Ca2+]i) of EA.hy926 cells was assessed by laser scanning confocal microscopy.Results:1) CHR inhibits the endothelial hyperpermeability induced by TNF-a. Compared with the control group, the permeability of EA.hy926 cells increased significantly in TNF-a group (1.19±0.09 vs 1.77±0.20, P<0.05). CHR (10 nmol/L,100 nmol/L and 1000 nmol/L) could inhibit the hyperpermeability induced by TNF-a in EA.hy926 cells; in that, CHR(10 nmol/L and 100 nmol/L) decreased significantly the hyperpermeability of EA.hy926 cells induced by TNF-a (1.77±0.20 vs 1.32±0.13, P<0.05; 1.77±0.20 vs 1.24±0.18, P<0.05). CHR (1000 nmol/L) decreased the hyperpermeability of EA.hy926 cells induced by TNF-a, but there was no statistically significant difference (1.77±0.20 vs 1.41±0.26, P>0.1).2) CHR inhibits the expression and distribution change of permeability-related protein in endothelial cells induced by TNF-a. Compared with control group, the F-actin of the EA.hy926 cells redistributed and formatted more stress fibers induced by TNF-a; CHR obviously reduced the stress fibers formatted and redistributed in EA.hy926 cells.3) CHR inhibits the extracellular of calcium in endothelial cells induced by TNF-a. The exposition to CHR (10 nmol/L,100 nmol/L and 1000 nmol/L) couldn’t induce a significantly different intracellular Ca2+ concentration in EA.hy926 cells. Exposition to TNF-a (22 ng/ml) could evoke a rapid Ca2+entry in EA.hy926 cells, the comparison of the Ca2+ fluorescence intensity at different time points showed that Ft40 and Ftioo had significant difference compared to initial fluorescence intensity Ft30 (41.50±3.79 vs 56.19±3.08, P<0.01; 41.50±3.79 vs 53.46±4.54, P<0.01), CHR (10 nmol/L,100 nmol/L and 1000 nmol/L) could inhibit TNF-a induced rapid Ca2+ influx.Conclusions:CHR could inhibit the TNF-a induced hyperpermeability in EA.hy926 cells, probably by blocking TNF-a evoking extracellular Ca2+ entry.