Gabor Than Award Lecture 2008: Pre-eclampsia – From Placental Oxidative Stress to Maternal Endothelial Dysfunction

An overview of redox generation and clearance. The superoxide anion (O₂⁻) is formed by the univalent reduction of triplet-state molecular oxygen (³O₂). This process is regulated enzymatically by NAD(P)H oxidases and xanthine oxidase or non-enzymatically by redox-reactive compounds (e.g. semi-ubiquinone compound). O₂⁻ is detoxified by manganese (if in mitochondria) or copper/zinc (if in cytosol) superoxide dismutase enzyme (MnSOD or Cu/ZnSOD). SOD converts superoxide into hydrogen peroxide (H₂O₂), which can subsequently be converted into water by the enzymes catalase or glutathione peroxidase. Alternatively, H₂O₂ can be converted into the highly reactive hydroxyl radical (OH) in the presence of reduced transition metals via the Fe²⁺-dependent Fenton reaction. GSH – glutathione; GSSG – glutathione disulphide.

An overview of redox-regulated protein kinase signalling in the route to apoptosis. Cells are equipped with various intracellular signal transduction systems, including protein kinases that facilitate transmission of physiological ROS-mediated signals. TNF-α induces ROS production via mitochondria. ASK1 is an upstream regulator of the stress-activated MAPK cascades and has been shown to play a critical role in apoptosis. TRX is a key protein that can act as a negative (ASK1) or, in combination with Ref, as a positive regulator. p38 and SAPK MAPKs promote apoptosis through activation of caspase-3. AKT, a key target of PI3-K, inactivates pro-apoptotic proteins (GSK-3) and activates transcription factors, which target anti-apoptotic proteins. Under oxidative stress, this pathway is activated by oxidative inactivation of phosphatases (e.g. PTEN), allowing constitutive activation of tyrosine kinase receptor and PI3-K. AKT acts in concert with ERK, SAPK and p38 to signal the intracellular apoptotic machinery for a full execution of apoptosis. Expression or product activity of some specific transcription factor genes are regulated in response to oxidative stress, which in turn determines the transcription level of genes that control proliferation, differentiation and cell death. Positive (e.g. MAPK-mediated) and negative (e.g. anti-oxidant response) regulatory circuits determine the final levels of ROS.

Stress pathways regulated by labour in vivo and hypoxia–reoxygenation and H₂O₂ in vitro. (A) Lysates from placentae delivered by caesarean section (CS) or vaginally with labour (L) were immunoblotted and analyzed with anti-cleaved caspase-3, anti-cleaved caspase-9 and anti-cleaved PARP antibodies. All blots were re-probed with anti-β-actin to normalise gel loading and normalised results (±SE) are plotted, expressing caesarean controls as 100%. (B) Protein lysates from placentae cultured under H/R or H₂O₂ (1mM) in the presence or absence of vitamins C and E (V) for 7h (n=6) were analysed for ASK1. Different letters indicate groups that are significantly different using the PLSD test with P<0.05.

The effect of sulfasalazine on H/R-induced expression of NF-κB, COX-2, sFlt-1 and cleaved caspase-3. Protein lysates from placentae cultured under H/R in the presence or absence of 0.5mM sulfasalazine (+0.5SS) or 2.5mM sulfasalazine (+2.5SS) for 7h (A) or 16h (B) were analysed for NF-κB pathway activation (A) or COX-2, sFlt-1 and cleaved caspase-3 (CC-3) protein expression (B). Different letters indicate groups that are significantly different using the PLSD test with P<0.05.

The effect of in vitro H/R on activation of sFlt, ADMA and on eNOS. Protein lysates from placentae cultured under H/R in the presence or absence of vitamins C and E (V) or PD169316 (p38i) for 7h were analysed with: (A) anti-sFlt, anti-HIF-1α, anti-VEGF; or (B) anti-P-eNOS (B), and anti-eNOS. (C) Secretion of ADMA into supernatants was measured by ELISA and normalised against wet tissue weight. (D) Immunostaining for endothelin-1 (green) and HNE (red) localised both markers principally to the syncytiotrophoblast. Different letters indicate groups that are significantly different using the PLSD test with P<0.05.

A proposed overview summarising how placental-derived factors mediate the symptoms of pre-eclampsia. Deficient conversion of spiral arteries leads to fluctuations in O₂ concentration and triggers ischaemia-reperfusion type injury of the placenta and consequent oxidative stress and ER stress. ROS activate various stress pathways, including pro-apoptotic p38 and SAPK/JNK MAPK pathways and inflammatory NF-κB pathway. These pathways promote increased shedding of microparticles, anti-angionenic factors and inflammatory cytokines, which lead to development of the peripheral maternal symptoms.