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


METHODS
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 micromol/L (interleukin-8, 1 pg/mL), 3.14+/-2.45 micromol/L (interleukin-8, 5 pg/ml), and 9.08+/-2.69 micromol/L (interleukin-8, 25 pg/mL; P<.01; compared with 0.77+/-0.47 micromol/L [control endothelial cells]). Horseradish peroxidase leakage in preeclamptic endothelial cells was 6.20+/-2.19 micromol/L, 8.44+/-85 micromol/L, and 15.79+/-2.06 micromol/L (P<.05) compared with 5.23+/-1.28 micromol/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).


CONCLUSIONS
Increased endothelial permeability may be associated with decreased endothelial nitric oxide synthase expression and activity in endothelial cells from preeclampsia.
Wang
Yuping
Y
Department of Obstetrics and Gynecology, Louisiana State University Health Sciences Center, Shreveport, LA, USA. ywang1@lsuhsc.edu
Gu
Yang
Y
Zhang
Yanping
Y
Lewis
David F
DF
D016022:Case-Control Studies; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000008:administration & dosage; D005260:Female; D006735:Horseradish Peroxidase / Q000493:pharmacokinetics; D006801:Humans; D016209:Interleukin-8 / Q000008:administration & dosage; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D009994:Osmolar Concentration; D011225:Pre-Eclampsia / Q000378:metabolism*; D011247:Pregnancy; D012333:RNA, Messenger / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism; D019323:omega-N-Methylarginine / Q000008:administration & dosage
D004791:Enzyme Inhibitors; D016209:Interleukin-8; D012333:RNA, Messenger; D019323:omega-N-Methylarginine; D006735:Horseradish Peroxidase; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III