Oxidative Stress and Release of Cell-free Mitochondrial DNA from Trophoblast Cells
0000-0003-3784-9190 (Gardner, Jennifer)
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Cell free mitochondrial DNA (mtDNA) is an indicator of cellular stress and systemic inflammation. These properties are accentuated when mtDNA undergoes oxidative damage. In addition, toll-like receptor 9 (TLR9), a receptor of the innate immune system, is activated by mtDNA. Inflammation, oxidative stress, and cell death are characteristics of placental ischemia, a common feature of preeclampsia. Recent work from our lab has shown dysregulation of circulating cell-free mtDNA in pregnancies with preeclampsia and association of this dysregulation with preeclampsia diagnosis. However, mechanisms underlying the release of mtDNA remain unclear. We hypothesized that human trophoblast cells exposed to oxidative stress via antimycin A, an inhibitor of complex III of the electron transport chain, would induce release of mtDNA via cell death-dependent mechanisms, leading to increased TLR9 activation. BeWo cells (ATCC? CCL-98) were treated with increasing concentrations of antimycin A (10, 50, 100, 320 µM) and vehicle (ethanol, 0.16% v/v) for 4 hours. Supernatants were collected and snap frozen in liquid nitrogen. Absolute real-time qPCR quantification with TaqMan™ probes and chemistry was used to quantify cell-free mtDNA (amplification target: MT-ND5 gene) and nuclear DNA (nDNA). Flow cytometry was used to assess the activation of cell death mechanisms in response to oxidative stress. To determine TLR-9-associated immunostimulatory potency of cell culture supernatants, we used an engineered cell line of human embryonic kidney 293 cells transfected with a human TLR-9 gene (HEK-BlueTM hTLR9). Exposure of trophoblast cells to antimycin A did not induce the release of mtDNA (p>0.05) or nDNA (p>0.05). Similarly, there were no differences in TLR9 activation between groups (p>0.28). Antimycin A (320 µM) reduced cell viability (Vehicle: 64.44 ± 5.46% vs Antimycin A: 18.14 ± 5.78%, p< 0.05) and increased necrosis (Vehicle: 10.39 ± 3.11% vs Antimycin A (100, 320 µM): 30.51 ± 4.43%, 40.16 ± 5.08%, P< 0.05), while apoptosis levels remained unchanged (P>0.1). Activation of oxidative stress pathways, via inhibition of complex III of the electron transport chain, leads to cell death, but does not affect release of mtDNA. These data suggest other cellular mechanisms, such as mitophagy or activation of antioxidant pathways, may serve a cytoprotective role against oxidative stressors in trophoblast cells. This study extends our pre-clinical knowledge about the links between placental oxidative stress and immunogenic factors in trophoblast cells. These findings may contribute to development of novel therapeutic targets for treatment of maternal cardiovascular dysfunction in preeclampsia.