Evidence of endothelial dysfunction in preeclampsia: decreased endothelial nitric oxide synthase expression is associated with increased cell permeability in endothelial cells from preeclampsia

Abstract

Objective The purposes of this study were to examine endothelial nitric oxide synthase expression in endothelial cells and to determine whether the inhibition of endothelial nitric oxide synthase could impair endothelial barrier function in preeclampsia. Study design Messenger RNA and protein expression for endothelial nitric oxide synthase were examined in endothelial cells that were isolated from normal and preeclamptic pregnancies. Endothelial monolayer permeable response to interleukin-8 stimulation was determined. Normal endothelial cells that were treated with nitric oxide inhibitor were used to test the association of endothelial nitric oxide synthase and endothelial barrier function. Messenger RNA expression for endothelial nitric oxide synthase was determined by reverse transcription–polymerase chain reaction, and protein expression was determined by Western blot analysis. Endothelial permeability was measured by horseradish peroxidase leakage through endothelial cell filters. Interleukin-8 production was measured by enzyme-linked immunosorbent assay. Data were presented as mean±SE and analyzed by analysis of variance or nonparametric Mann-Whitney test. Results Relative messenger RNA expression and protein expression for endothelial nitric oxide synthase were decreased significantly in endothelial cells from preeclampsia compared with cells from normal pregnancies (messenger RNA expression, 0.191±0.057 vs 0.508±0.061 [ P < .01]; protein expression, 0.225±0.08 vs 0.786±0.098 [ P<.01], respectively). Horseradish peroxidase leakage in normal endothelial cells was 0.30±0.26 μmol/L (interleukin-8, 1 pg/mL), 3.14±2.45 μmol/L (interleukin-8, 5 pg/ml), and 9.08±2.69 μmol/L (interleukin-8, 25 pg/mL; P<.01; compared with 0.77±0.47 μmol/L [control endothelial cells]). Horseradish peroxidase leakage in preeclamptic endothelial cells was 6.20±2.19 μmol/L, 8.44±85 μmol/L, and 15.79±2.06 μmol/L ( P<.05) compared with 5.23±1.28 μmol/L, respectively. The ratio of horseradish peroxidase leakage was >7-fold increase in normal endothelial cells, but only a 4-fold increase in preeclamptic endothelial cells in response to interleukin-8 stimulation at 25 pg/mL. The inhibition of endothelial nitric oxide synthase with N G-Monomethyl-L-arginine resulted in an increase in interleukin-8–induced endothelial cell permeability. No difference for interleukin-8 production was observed between normal and preeclamptic endothelial cells (1.15±0.21 ng/mg protein vs 1.29±0.23 ng/mg protein, P > .5). Conclusion Increased endothelial permeability may be associated with decreased endothelial nitric oxide synthase expression and activity in endothelial cells from preeclampsia.