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The rate of H2O2-induced F-actin formation increased linearly with increasing H2O2 concentrations, suggesting that increases in the concentration of H2O2 activate a signaling cascade that leads to actin reorganization. Administration of Ca2+-chelating agent BAPTA effectively blocked the formation of actin, suggesting that actin reorganization is Ca2+ dependent. The addition of VEGF, a potent permeability mediator, at low concentrations accelerated F-actin formation. This effect of VEGF was additive with that of H2O2, suggesting that VEGF and H2O2 act by an independent mechanism to promote the formation of F-actin. Both VEGF and H2O2 increased the amount of cytosolic free Ca2+, suggesting that both molecules induce F-actin formation by increasing [Ca2+]i in endothelial cells. The effect of H2O2 on [Ca2+]i was similar to that of VEGF, suggesting that H2O2 may act upstream of VEGF in a Ca2+-dependent signaling pathway.
Results: We have developed a high-throughput screening assay system that we used to identify and test small molecules that prevent H2O2-induced albumin permeability and that reduce the thrombin-induced permeability of HUVECs. We have identified a number of compounds that can prevent increases in endothelial permeability induced by either thrombin or H2O2. These compounds inhibit H2O2-induced elevation of intracellular free Ca2+, which is a major mediator of endothelial permeability. Inhibition of H2O2-induced elevation of intracellular Ca2+ was accompanied by a significant inhibition of thrombin-induced increase in endothelial permeability.
Endothelial cells, which normally remain tightly coupled at the cell-cell junctions, lose their intercellular adhesion and form intercellular gap when exposed to H2O2 and exogenous agonists (61, 105, 229). Decreased intracellular free Ca2+ concentration causes the decrease of actin polymerization, and thereby loss of the intercellular adhesion in the outer rim of the cell-cell contact area. This could be associated with the redistribution of junction proteins, such as occludin, ZO-1, and VE-cadherin. The redistribution of junction proteins probably contributes to the formation of gaps between the cells. 827ec27edc