Oxidative stress induces non-apoptotic cell death and release of placental derived mitochondrial DNA

Abstract

Introduction: The placenta, a metabolically active tissue, forms a critical interface between the developing fetus and mother. Stressors such as oxidative stress contribute to placental inflammation, cell death, and release of proinflammatory factors into the maternal circulation. Extracellular mitochondrial DNA (mtDNA) is a proinflammatory factor that can be found in blood circulation in response to cellular stress. Circulating cell-free mtDNA increases during healthy pregnancy and is dysregulated in pregnancies with preeclampsia. Mechanisms underlying the release of mtDNA into circulation from the placenta remain unclear. The objective of this study is to elucidate the relationship between oxidative stress, cell death, and the release of mtDNA from the placenta. We hypothesized that human placental cells exposed to oxidative stressors release mtDNA into the extracellular space via cell death-dependent mechanisms. Methods: BeWo cells (ATCC ® CCL-98), a model of placental trophoblast, were treated with the oxidative stressors, rotenone (0.8, 5, 10, 25, 50 μM) or antimycin A (10, 50, 100, 320 μM) and their respective vehicles for 4 hours. Supernatants were collected and the following experiments were performed: absolute real-time qPCR quantification with TaqMan™ probes and chemistry to quantify intracellular and extracellular mtDNA (amplification target: MT-ND5gene) and nuclear DNA (nDNA), assays to measure reactive oxygen species, flow cytometry to assess cell death mechanisms. Results: Exposure of placental cells to antimycin A induced the release of mtDNA (p<0.0001) and nDNA (p=0.024), whilst intracellular mtDNA copy number was unaffected (p=0.62). Antimycin A (320 μM) reduced cell viability (vehicle: 64.44 ± 5.46% vs antimycin A: 18.14 ± 5.78%, p=0.0005), increased non-apoptotic cell death (vehicle: 10.39± 3.11% vs antimycin A (100, 320 μM): 30.51 ± 4.43%, 40.16± 5.08%, p=0.0001), and did not affect apoptosis (p=0.11). In addition, there was no change in the release of mtDNA (p=0.43) or nDNA (p=0.82), cell viability (p=0.94), non-apoptotic cell death (p=0.52), or apoptosis (p=0.85) in cells treated with rotenone. Conclusions: Placental cells exposed to oxidative stressors undergo non-apoptotic cell death and release mtDNA. These responses are only observed at high concentrations of antimycin A, a pharmacological inhibitor of complex III of the electron transport chain (ETC), but not rotenone, an inhibitor of complex I of the ETC, suggesting the presence of cytoprotective or rescue mechanisms against oxidative stressors in the placenta. NIH R01 HL146562, AHA 22PRE-900431, AHA 22POST-903250, NIH T32 AG 020494 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.