Antioxidant superoxide dismutase attenuates increased endothelial permeability induced by platelet-activating factor

Abstract

To study the protective effects of the antioxidant superoxide dismutase (SOD) against platelet-activating factor (PAF)-induced endothelial permeability. Endothelial cells (ECs) were isolated from human umbilical veins from normal pregnancies. The first passage (P1) ECs were grown in polycarbonate transwell filters. Confluent ECs were incubated with PAF at concentrations of 2, 5, and 10 microgram/mL for 2 hours or pretreated with superoxide dismutase. Endothelial monolayer permeability was then measured by EC electrical resistance or by the leakage of horseradish peroxide (HRP) passing through filters. Endothelial junctional protein distribution and expression of VE-cadherin and occludin were determined by fluorescent staining of endothelial monolayer and by Western blot analysis. mRNA expressions for VE-cadherin and occludin were determined by reverse transcriptase-polymerase chain reaction. Data are expressed as Omega. cm(2) for electrical resistance and DeltaOD 470 nm for HRP assay and presented as mean +/- standard error of the mean. Analysis of variance was used for statistical analysis. A P value less than.05 was considered statistically significant. Endothelial cell electrical resistance was decreased and HRP leakage was increased in ECs treated with PAF. Intercellular gaps were formed at cell contact regions in ECs treated with PAF, as evaluated by staining of junctional protein VE-cadherin and occludin. The functional changes of the EC barrier and the formation of intercellular gaps induced by PAF were concentration dependent, which could be partially attenuated by pretreatment of ECs with SOD. Total cellular junctional protein expression and mRNA expression of VE-cadherin and occludin were not affected by PAF. Increased EC monolayer permeability induced by platelet-activating factor is associated with disorganization of EC junctional protein distribution of VE-cadherin and occludin. Superoxide dismutase partially attenuated the PAF-induced increased endothelial monolayer permeability, which suggests that oxidative stress might be involved in the process of PAF-induced disturbances of endothelial barrier function.