Molecular mechanism for change in permeability in brain microvascular endothelial cells induced by LPS.

Abstract

To investigate the molecular mechanism for change in permeability in brain microvascular endothelial cells (bEnd.3) induced by lipopolysaccharide (LPS). Monolayers of bEnd.3 were exposed to LPS, in the presence or absence of exoenzyme C3 transferase. We monitored the monolayer barrier integrity by transendothelial electrical resistance assay (TEER), activity of RhoA by pull down assay, NF-κB by luciferase reporter assay, and F-actin dynamic structure by Rhodamine-phalloidin staining. Incubation of monolayers with LPS caused substantial barrier hyperpermeability. Under the normal condition, the average TEER of bEnd.3 was (82.33±3.11) ω.cm², while it decreased apparently to (53.67±2.01) ω.cm² and (37.67±3.05) ω.cm² when the cells had been treated for 3 and 12 h with LPS (P<0.05). Such effects could be inhibited partly by pretreatment of RhoA inhibitor exoenzyme C3 transferase. LPS activated RhoA and NF-κB at 0.5 h. The C3 transferase could significantly reverse the NF-κB activation (P<0.05). The F-actin rearrangements displayed in a time-dependent manner and occurred originally after the stimulation of LPS for 3 h, which could be diluted by the pretreatment of C3 transferase as well. LPS induces the disruption of F-actin cytoskeleton and brain microvascular endothelial barrier integrity, in part, through RhoA and NF-κB activation. The mechanism underlying this pathophysiological effect of RhoA is to influence the disruption of the F-actin cytoskeleton by regulating NF-κB activities.