Gas6 Reduces Cellular Respiration and Increases Reactive Oxygen Species in Immortalized Human First Trimester Trophoblast Cells

Hypertension and proteinuria are two hallmarks of preeclampsia (PE), an obstetric pathology with an elusive etiology that affects 5–10% of pregnancies. At the cellular level, some indications of preeclampsia are decreased trophoblast invasion and increased anti‐angiogenic and cytokine production during placentation. As a protein, growth arrest‐specific 6 (Gas6) has been widely studied in the prevention of apoptosis and enhanced cell migration and invasion. Gas6 has been found to be increased during PE. Previously, we have demonstrated decreased trophoblast invasion with Gas6 treatment and increased blood pressure and proteinuria when rats are injected with Gas6. The effects of Gas6 can be reversed by inhibiting one of its ligands, AXL. Our objective was to determine the effects of Gas6 cellular respiration and reactive oxygen species (ROS) production in the immortalized human first trimester trophoblast cell line SW71. Cultured cells were treated separated into three groups (n=4): Gas6 (600 ng/mL), Gas6 and AXL inhibitor R428 (600 ng/mL), or control. We determined O 2 consumption at 37°C in permeabilized cells using the Oroboros O 2 K Oxygraph (Innsbruck, Austria) with MiR05 respiration buffer. Respiration was determined by using a substrate‐uncoupler‐inhibitor‐titration protocol examining function of complex I, oxidative phosphorylation, complex II and full electron transport system capacity. ROS generation was assessed using the Muse® Oxidative Stress Kit (EMD Millipore, Billerica, MA) as per manufacturer's instructions. Samples were run on the Muse® benchtop flow cytometer (EMD Millipore, Billerica, MA). Overall, our results demonstrated decreases in cellular respiration when cells were exposed to Gas6 when compared to control. We also observed an increase in ROS when cells were treated with Gas6 when compared to control. Both decreased respiration and increased ROS yield were reversed when cells were treated with the AXL inhibitor R428. These results suggest that the stress‐mediated response to the presence of Gas6, as seen in preeclampsia, could involve metabolic pathways that decrease cellular respiration and increase ROS species production.