Angiotensin-converting enzyme 2 regulates the placental response to repeated hypoxia-reoxygenation insult

Fetal growth restriction is linked to placental oxidative stress owing to defective placentation and hypoxia-reoxygenation (H/R). Angiotensin-converting enzyme 2 (ACE2), a key regulator of oxidative stress, is reduced in placentae of fetuses with growth restriction. We aimed to develop a physiologically relevant model of repeated H/R to determine the protective role of ACE2 during repeated H/R insult. Human term placental explants (n = 8 per group) were cultured for 24 h in normoxia (8% O₂), non-physiological H/R [6 h cycles of 1% (hypoxia chamber) and 8% O₂ (incubator)] or our novel physiological H/R model (6 h cycles of 1% and 8% O₂ in a single hypoxia chamber), with or without recombinant human (rh)ACE2. Physiological H/R, in contrast to non-physiological H/R, decreased membrane-bound ACE2 protein compared with normoxia. Physiological H/R also increased NADPH oxidase 5 (NOX5) mRNA and protein, NOX4 protein and xanthine dehydrogenase (XDH) mRNA compared with normoxia. Physiological H/R increased levels of antioxidants superoxide dismutase 1 (SOD1) mRNA, heme oxygenase 1 (HO-1) mRNA and protein, and glutathione reductase (GSR), nuclear factor erythroid 2-related factor (Nrf2) and NADPH quinone oxidoreductase 1 (NQO1) mRNA above normoxia. Conversely, physiological H/R reduced SOD, CAT and GPx activity below normoxia. Non-physiological H/R had no effect on ROS or antioxidant enzymes except for increasing GSR mRNA compared with normoxia. The rhACE2 reduced NOX5 mRNA and NOX4 and NOX5 protein and increased CAT and GPx activities compared with physiological H/R alone. Using a novel repeated H/R model, we show that repeated H/R downregulates ACE2 protein and induces placental oxidative stress, which can be mitigated by rhACE2 in vitro. KEY POINTS: A physiologically relevant repeated hypoxia-reoxygenation (H/R) model was established using human placental explants that show upregulated NOX5, NOX4 and XDH, driving the generation of reactive oxygen species. Repeated H/R insult reduces membrane-bound ACE2 protein, contributing to oxidative stress in placental tissue. The mRNA and protein levels of key antioxidants (SOD1, HO-1, GSR, Nrf2 and NQO1) were elevated, but paradoxically, enzymatic activities (SOD, CAT and GPx) were reduced. Recombinant human ACE2 supplementation reversed oxidative markers and restored antioxidant enzyme activities, thereby mitigating H/R-induced placental dysfunction. Our findings enhance understanding of placental oxidative regulation and offer new therapeutic direction for fetal growth restriction.