Oxidative Stress Drives ECM Shedding in Placental Trophoblasts
Authors: Eddy Adrian C. , Chapman Heather , George Eric M. 
Published: November 2023

Preeclampsia (PE) is a pregnancy disorder characterized by new onset of hypertension, and the development of other clinical symptoms such as proteinuria, cerebrovascular dysfunction, and liver/kidney dysfunction. Affecting approximately five percent of pregnancies, PE is the primary cause of maternal and fetal morbidity. Though the precise etiology remains unclear, placental hypoxia and ischemia is believed to be the central cause, after which the ischemic placenta releases pro‐hypertensive factors into the maternal circulation. One of the primary secreted drivers of the associated maternal hypertension is believed to be the anti‐angiogenic protein sFlt‐1. Interestingly, placental oxidative stress has also been implicated in the development of PE, but the link between placental ROS and maternal hypertension is not clear. Our lab recently demonstrated that release of the pro‐hypertensive protein sFlt‐1 is reliant on shedding of the cellular extracellular matrix (ECM) protein Syndecan‐1 (SDC‐1), as sFlt‐1 is normally immobilized by binding to SDC‐1 heparan side chains under basal conditions. Here, we test the hypothesis that hypoxia‐dependent syndecan shedding is dependent on increased oxidative stress in placental cells, using an immortalized human placental trophoblast cell line (BeWo). Two plates of cells, to be placed in 8% oxygen or in 1% oxygen (approximate oxygen concentration in a healthy and ischemia placenta respectively), were divided into four groups: control, 1mM tempol, 100μM apocynin, and a combined treatment of tempol and apocynin (n=4 per group). The cells were exposed for 24 hours before the media collected and an ELISA performed against human Syndecan‐1. The 1% control showed a hypoxic response with a 67% increase in SDC‐1 versus the 8% control (281.5 ± 5.52 pg/mL vs 168.3 ± 7.63 pg/mL; p<0.005). Of the treated groups, the apocynin treated cells showed a 34% decrease in syndecan shedding versus the control in the cells exposed to 1% oxygen (184.8 ±4.07 pg/mL vs 281.5 ± 5.52 pg/mL; p<0.005); the cells exposed to both tempol and apocynin similarly showed a 36% decrease (180.3 ± 4.48 pg/mL vs 281.5 ± 5.52 pg/mL; p<0.005). Because the mechanism of apocynin has been debated as being either a NADPH oxidase (NOX) inhibitor or a peroxide scavenger, we performed lucigenin chemiluminescence for superoxide and amplex red fluorescence for peroxide on the 8% oxygen cells which were untreated, treated with tempol, and treated with apocynin. While noting no difference in superoxide detection by lucigenin, amplex red fluorescence showed a decrease in peroxide of approximately 28% in the tempol treated cells (17054 ± 580.5 AU vs 23789 ±1181 AU; p<0.005) and a decrease of nearly 51% in the apocynin treated cells (11713 ± 202.9 AU vs 23789 ± 1181 AU; p<0.005). Using an inhibitor of Nox4, which exclusively produces peroxide, we found that cells exposed to 1% oxygen showed a 20% decrease in syndecan shedding versus their control counterparts (245.0 ± 13.57 pg/mL vs 306.0 ± 17.42 pg/mL; p<0.05). Though the mechanism is still uncertain, we can conclude that ROS, specifically peroxide produced by Nox4, may play a role in hypoxic syndecan shedding, and thus release of sFlt‐1, and this pathway may be an object of future study in the development of preeclampsia.

Support or Funding Information

NIH P01HL51971, P20GM104357, R00HL116774; American Heart Association GRNT31460000