We determined placental tissue levels, production rates, and secretion rates of isoprostanes for placentas obtained from women with normal pregnancies and women with preeclampsia, a hypertensive disorder of pregnancy. Isoprostanes are markers of oxidative stress that exert biological actions such as vasoconstriction. Placental tissue was rinsed and immediately frozen in liquid nitrogen to determine tissue levels of total and free isoprostane. Placental tissue pieces were also incubated in serum-free DMEM for 48 h at 37 degrees C in 95% air/5% CO(2) to determine production rates. Isolated placental cotyledons were perfused for the determination of secretion rates. All samples were analyzed by EIA for isoprostane using an antibody specific for 8-Iso-PGF(2) (15-F(2t)-IsoP). In addition, medium samples were analyzed for malondialdehyde (MDA), a breakdown product of lipid peroxidation. We found that tissue levels of free isoprostane and total isoprostane (free plus esterified forms) were significantly higher for preeclamptic placentas than for normal placentas. Concentrations of isoprostane and MDA in the medium increased progressively during 48 h of incubation of placental explants. At 48 h of incubation, the mean concentrations of both isoprostane and MDA were significantly higher for the placentas from preeclamptic women than for the placentas from normal pregnant women. Concentrations of MDA were highly correlated with those of isoprostane. Induction of oxidative stress with xanthine plus xanthine oxidase increased placental production of isoprostane by normal tissue to a level similar to that of preeclamptic tissue. Placental secretion of isoprostane was eightfold greater toward the maternal side of the placenta than toward the fetal side, and was increased sixfold on the maternal side and twofold on the fetal side by inducing oxidative stress with t-butyl hydroperoxide. This study presents new information that isoprostanes are formed and secreted by the human placenta and provides convincing evidence that oxidative stress and lipid peroxidation are abnormally increased in placentas of preeclamptic women.

Objective Placentas are oxidatively stressed during preeclampsia and produce more TNFα and more thromboxane (TX) than normal. Oxidative stress may cause these abnormalities by activating NF-κB. We measured levels of activated NF-κB in normal and preeclamptic placentas and determined whether oxidative stress activates NF-κB in a trophoblast-like cell line. Methods We used immunohistochemistry to determine the percentage of the total tissue area that stained for the p65 subunit of NF-κB in placentas obtained from normal and preeclamptic pregnancies. In a second set of experiments, we used a reporter plasmid bearing the NF-κB binding site and transfected it into trophoblast-like cells. The cells were incubated with medium control, linoleic acid (LA), an oxidizing solution (Ox), or Ox enriched with linoleic acid (OxLA), TNFα, or OxLA plus TNFα for 20 hours. Cell lysates were analyzed using a dual luciferase assay kit. Results Placentas obtained from women with preeclampsia showed nearly a 10-fold increase in the extent of area stained for activated NF-κB as compared to normal placentas. In cell culture experiments, Ox and OxLA induced a 3-fold increase in NF-κB activation as compared to medium control or LA. TNFα induced a 3-fold increase in NF-κB activation. The combination of TNFα with OxLA caused a 10-fold increase in NF-κB activation. Conclusions Placental NF-κB is activated nearly 10-fold in preeclampsia. Oxidative stress causes NF-κB activation in a trophoblast-like cell line, which is enhanced by TNFα. These data suggest that oxidative stress is likely an important in vivo activator of placental NF-κB in preeclampsia.

We determined placental tissue levels, production rates, and secretion rates of isoprostanes for placentas obtained from women with normal pregnancies and women with preeclampsia, a hypertensive disorder of pregnancy. Isoprostanes are markers of oxidative stress that exert biological actions such as vasoconstriction. Placental tissue was rinsed and immediately frozen in liquid nitrogen to determine tissue levels of total and free isoprostane. Placental tissue pieces were also incubated in serum-free DMEM for 48 h at 37 degrees C in 95% air/5% CO(2) to determine production rates. Isolated placental cotyledons were perfused for the determination of secretion rates. All samples were analyzed by EIA for isoprostane using an antibody specific for 8-Iso-PGF(2) (15-F(2t)-IsoP). In addition, medium samples were analyzed for malondialdehyde (MDA), a breakdown product of lipid peroxidation. We found that tissue levels of free isoprostane and total isoprostane (free plus esterified forms) were significantly higher for preeclamptic placentas than for normal placentas. Concentrations of isoprostane and MDA in the medium increased progressively during 48 h of incubation of placental explants. At 48 h of incubation, the mean concentrations of both isoprostane and MDA were significantly higher for the placentas from preeclamptic women than for the placentas from normal pregnant women. Concentrations of MDA were highly correlated with those of isoprostane. Induction of oxidative stress with xanthine plus xanthine oxidase increased placental production of isoprostane by normal tissue to a level similar to that of preeclamptic tissue. Placental secretion of isoprostane was eightfold greater toward the maternal side of the placenta than toward the fetal side, and was increased sixfold on the maternal side and twofold on the fetal side by inducing oxidative stress with t-butyl hydroperoxide. This study presents new information that isoprostanes are formed and secreted by the human placenta and provides convincing evidence that oxidative stress and lipid peroxidation are abnormally increased in placentas of preeclamptic women.

These data mimic the changes in placental SOD, GSH-Px, catalase, TX and lipid peroxidation that occur in preeclampsia suggesting that maternal hyperlipidemia and increased iron levels may be responsible for placental oxidative stress and abnormalities in antioxidants and thromboxane.

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