Effects of peroxynitrite and superoxide radicals on endothelial monolayer permeability: potential role of peroxynitrite in preeclampsia.

Objective: Increased endothelial permeability is associated with increased oxidative stress in the maternal vasculature in women with preeclampsia. This study was to determine if oxidative stress elicited by peroxynitrite could lead to an increase in endothelial permeability.

Methods: Endothelial oxidative stress was produced by adding 3-morpholinosydnonimine (SIN-1, a peroxynitrite generator) to the cell culture. Confluent endothelial cells (ECs) grown in cell culture inserts were treated with SIN-1 at a concentration of 0.5 mM alone or in combination with MnTMPyP (a peroxynitrite scavenger) or superoxide dismutase (SOD). EC permeability was determined by measuring EC electrical resistance (ER) and horseradish peroxide (HRP) leakage. Data are presented as means +/- SE and analyzed by analysis of variance (ANOVA). Junctional protein expression and distribution for vascular endothelial (VE)-cadherin, occludin, and phosphorylated focal adhesion kinase (FAK) at tyrosine 397 [pY397] were examined by fluorescent staining of ECs.

Results: First, ER was significantly reduced and HRP leakage was significantly increased in ECs treated with SIN-1 compared to those in control cells, ER: 26.97 +/- 1.41 versus 42.27 +/- 0.40 Omega.cm2, P <.01; HRP: 0.26 +/- 0.07 versus 0.02 +/- 0.01 OD 470 nm, P <.01, respectively. Second, cells treated with SIN-1 showed formation of gaps and disorganized VE-cadherin and occludin distribution at cell contact regions. FAK[pY397] expression was completely lost in cells treated with SIN-1. Finally, these functional and morphologic changes in ECs induced by SIN-1 were blocked in cells pretreated with MnTMPyP and SOD.

Conclusions: Disorganization of junctional proteins and dephosphorylation of FAK[pY397] may account for the increased endothelial permeability induced by oxidative stress associated with preeclampsia.