| Background and Objective In the multiple organ failure and systemic infammatory response syndrome, failure of the blood vessel wall to retain fluid and plasma solutes in the vascular space is a key character of inflammation. Endothelial barrier function is determined by the area available for fluid and solute exchange, and actin cytoskeleton is required for barrier maintenance, and filamentous F-actin" disintegration initates the barrier failure. Endothelial cell permeability induced by thrombin and LPS is accompanied by actin stress fibres assembility and intercellular barrier function, and correlate this with their effects on F-actin organization.It has been recently confirmed that Rho GTPases family paly an important role in regulation of endothelial permeability and Rho GTPases control cytoskeletal reorganization to reduce the endothelial barrier in response to external stimulition.In this study,we cultured the ECV304 cells in Transwell chamber to investigated the effect of LPS and thrombin on the endothelial permeability of endothelial cell mono layers RhoA gene expression and the change of F-actin and to study the influence of Y-27632,a special inhibitor of Rho activated kinase inhibitor,on the endothelial permeability and the change of cytoskeleton. We purpose that wether the Rho GTPase pathway is critical for the activation of endothelial cells by LPS and thrombin and can be a new target to prevent the change of endothelial cell barrier function.Methods (l)The effect of LPS and thrombin on the endothelial permeability of the ECV304 cells monolayers were investigated by measuring paracellular flux of HRP.Medium was then replaced with fresh serum free DMEM in the presence or absence of Y-27632 and the incubation was continued for 60 minutes.The HRP content of the samples was evaluated spectrophotometrically.(2)F-actin was stained by fluorescene isothiocyanate-conjugated phalloidin and examined the change ofendothelial cell cytoskeleyon induced by LPS and thrombin in the presence or absence of Y-27632 using fluorescent microscope.(3)The expression of RhoA mRNA in ECV304 cells was examined were determined by RT-PCR method.Results (1) After endothelial cell stimulation with thrombin and LPS, endothelial cell permeability was previously increased compared with the control group in a dose-and-time dependent manner (p<0.05) .Thrombin induced a rapid and transient(5min) increase and lasted for >2 hour in the barrier function, as evidenced by a increase in the endothelial cell permeability.LPS by itself induced a prolonged barrier dysfunction(>2h).The increase in permeability was completely inhibited by Rho kinase inhibitor,Y-27632 (p<0.05) .(2) Under basal conditions, F-actin in ECV304 cells was arranged into a fine cortical network with a dense peripheral band at the cell boundaries; occasionally, stress fibers were observed. The cells pretreated with LPS and thrombin showed F-actin bundles in stress fibers and some gaps became visible at the cell-cell contact areas.The ability of Y-27632 to prevent loss of tight junction in thrombin and LPS stimulated cells provides an explanation for their inhibitory effect on the increased endothelial permeability. (3) Thrombin and LPS enhanced ECV304 cells RhoA mRNA expression in a dose-dependent manner (P< "0.05).Conclusion During thrombin and LPS induced the increase of permeability and the change of cytoskeleton in endothelial cell, Rho kinase signal pathway is activated and RhoA gene expression is enhanced; inhibition of the pathway can change the endothelial cell permeability, which suggests that the Rho GTPase pathway may play an important role in thrombin and LPS induced endothelial cell permeability and inhibition of this novel cell signaling pathway of thrombin and LPS could be relevent for the prevention of the systemic infammatory response syndrome. |
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