Evaluating oxidative stress targeting treatments in in vitro models of placental stress relevant to preeclampsia

Dinara Afrose ¹ Matt Johansen ¹ Valentina Nikolic ² Natasa Orlic ³ Zeljko Mikovic ³ Milan Stefanovic ⁴ Zoran Cakic ⁵ Philip Michael Hansbro ¹ Lana McClements ^1*

• ¹ University of Technology Sydney, Sydney, Australia
• ² University of Niš, Niš, Southern and Eastern Serbia, Serbia
• ³ Gynecology and Obstetrics Clinic Narodni front, Belgarde, Serbia
• ⁴ Faculty of Medicine, University of Niš, Niš, Serbia
• ⁵ General Hospital Leskovac, Leskovac, Serbia

BackgroundPreeclampsia is a complex pregnancy disorder characterized by the new onset of hypertension and organ dysfunction, often leading to significant maternal and fetal morbidity and mortality. Placental dysfunction is a hallmark feature of preeclampsia, which is often caused by inappropriate trophoblast cell function due to oxidative stress, inflammation and/or pathological hypoxia. This study explores the role of oxidative stress in trophoblast cell-based models mimicking the preeclamptic placenta and evaluates potential therapeutic strategies targeting these mechanisms.MethodsUric acid (UA) and malondialdehyde (MDA) concentrations were measured in human plasma from women with preeclampsia (n=24) or normotensive controls (n=14) using colorimetric assays. Custom-made first trimester trophoblast cell line, ACH-3P, was exposed to various preeclampsia-like stimuli including hypoxia mimetic (dimethyloxalylglycine or DMOG, 1mM), inflammation (tumour necrosis factor or TNF-α, 10ng/ml) or mitochondria dysfunction agent, (Rhodamne-6G or Rho-6G, 1 μg/ml), ± aspirin (0.5mM), metformin (0.5mM), AD-01 (100nM) or resveratrol (15 μM), for 48 h. Following treatments, UA/MDA, proliferation assay (MTT), wound scratch and cytometric bead, assays, were performed.ResultsOverall, MDA plasma concentration was increased in the preeclampsia group compared to healthy controls (p<0.001) whereas UA showed a trend towards an increase (p=0.06); when adjusted for differences in gestational age at blood sampling, MDA remained (p<0.001) whereas UA became (p=0.03) significantly correlated with preeclampsia. Our 2D first trimester trophoblast cell-based in vitro model of placental stress in preeclampsia, mimicked the increase in UA concentration following treatment with DMOG (p<0.0001), TNF-α (p<0.05) or Rho-6G (p<0.001) whereas MDA cell concentration increased only in the presence of DMOG (p<0.0001) or Rho-6G (p<0.001). Metformin was able to abrogate Rho-6G- (p<0.0001) or TNF-α- (p<0.01) induced increase in UA, or DMOG-induced increase in MDA (p<0.0001). AD-01 abrogated UA or MDA increase in the presence of TNF-α (p<0.001) or Rho-6G (p<0.001), respectively. The preeclampsia-like stimuli also mimicked adverse impact on trophoblast cell proliferation, migration and inflammation, most of which were restored with either aspirin, metformin, resveratrol, or AD-01 (p<0.05).ConclusionsOur 2D in vitro models recapitulate the response of the first trimester trophoblast cells to preeclampsia-like stresses, modelling inappropriate placental development, and demonstrate therapeutic potential of repurposed treatments.