Women’s Health
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21642
Browse
Browsing Women’s Health by Author "Cushen, Spencer"
Now showing 1 - 2 of 2
- Results Per Page
- Sort Options
Item Mitochondrial oxidative stress and extrusion of mitochondrial DNA from endothelial cells: implications for maternal endothelial dysfunction in preeclampsia(2019-03-05) Cushen, Spencer; Phillips, Nicole; Goulopoulou, Styliani; Nguyen, RyanPurpose: Preeclampsia is one of the leading causes of maternal mortality during pregnancy and a risk factor of cardiovascular disease for the mother later in life. Hypertension and endothelial dysfunction are common characteristics of the maternal syndrome in preeclampsia, and oxidative stress is considered a pathogenic mediator of these maternal features. Mitochondria are the primary cellular producers of reactive oxygen species (ROS) and overproduction of mitochondrial ROS (mtROS) is detrimental to cellular processes, often leading to cell death. Mitochondrial DNA (mtDNA) has pro-inflammatory properties when released from dying cells into the extracellular space and its concentrations are increased in women with preeclampsia. The objective of this study was to determine the effects of mtROS in mtDNA release into the extracellular space. We hypothesize that inhibition of mitochondrial transport chain results in extrusion of mtDNA from vascular endothelial cells. Also, inhibition of Complex III causes a greater release of mtDNA compared to inhibition of Complex I. Methods: Human umbilical vein endothelial cells (HUVEC) were grown to 80-90% confluency before being treated with a mitochondrial complex I inhibitor (Rotenone: 5, 10, 25 mM – 4h) and mitochondrial complex III inhibitor (Antimycin A: 10, 50, 100 mM – 4h). After treatment, the cell media supernatant was collected and stored in -80 °C until further mtDNA quantification. mtDNA was isolated using the Mag-Bind Blood & Tissue DNA HDQ 96 Kit and quantified using the TaqMan chemistry-based method of absolute qPCR. Results: HUVEC cells treated with rotenone, regardless of dose, had no effect on concentrations of extracellular mtDNA (Figure A; One-way ANOVA followed by Sidak’s post-hoc test). Concentrations of mtDNA increased in HUVECs treated with 100 mM of Antimycin A (Figure B; One-way ANOVA followed by Sidak’s post-hoc test). Lower concentrations of Antimycin A had no effect on concentrations of extracellular mtDNA (Figure B). Conclusions: Inhibition of mitochondrial respiratory chain complex III, but not inhibition of complex I, results in extrusion of mtDNA. The increase in mtDNA released from dysfunctional cells may contribute to the increased circulating mtDNA concentrations seen in pregnancies with maternal endothelial dysfunction, such as pregnancies with preeclampsia.Item Placental Exposure to Hypoxia and Oxidative Stress Causes Mitochondrial DNA Release into the Extracellular Space(2019-03-05) Osikoya, Oluwatobiloba; Blessing, Alexandra; Phillips, Nicole; Goulopoulou, Styliani; Cushen, SpencerPurpose In preeclampsia, a severe hypertensive disorder of pregnancy, placentae experience reduced perfusion, increased cell death, and oxidative stress. Also, there is an increase in circulating cell-free mitochondrial DNA (mtDNA) in the maternal blood. The main objective of this study was to determine the role of hypoxia and oxidative stress in mtDNA release from placental cells, and to examine the effects of soluble factors from hypoxia-exposed placentae on vascular reactivity. To address this objective, the following hypotheses will be tested: a) Exposure to hypoxia and oxidative stress will result in mtDNA release via cell-death dependent mechanisms in human trophoblast cells. b) Soluble factors from hypoxia-exposed placentae will result in reduced vasodilation in rat maternal arteries. Methods To examine the effects of preeclampsia-related placental stressors on mtDNA release, we treated human trophoblast cells (BeWo cell line) with: 1) hypoxia (1% O2) vs. normoxia (21% O2) for 15 h, or 2) a mitochondrial complex I inhibitor (Rotenone, 10 μM) vs. vehicle for 4 h. mtDNA in cell culture supernatant was measured using absolute qPCR and cell death was quantified using flow cytometry. To test the effects of hypoxic placenta-derived factors on maternal vascular function, we used mesenteric arteries and placenta-conditioned media (PLmedia) from pregnant rats. Placentae were incubated in physiological salt solution (37oC) for 3 h in either 1% or 21% O2, while arteries were mounted on a wire myograph and underwent a baseline [(-) PLmedia] concentration-response curve (CRC) to acetylcholine (ACh, 10-9 – 3x10-5 M) followed by 30-min incubation with PLmedia, after which the CRC was repeated. Results Exposure of trophoblast cells to rotenone resulted in cell death (Vehicle: 28.17 ± 2.67% vs. Rotenone: 48.43 ± 1.22%, n = 3, P = 0.002) and mtDNA release (Vehicle: 1.69 ± 0.12 ng/uL vs. Rotenone: 2.39 ± 0.10 ng/uL, n = 5, P = 0.002). Hypoxia did not induce trophoblast cell death (Normoxia: 24.7 ± 0.50% vs. Hypoxia: 24.25 ± 0.45%, n = 2, P = 0.6), but increased release of mtDNA (Normoxia: 14.22 ± 1.20 pg/uL vs. Hypoxia: 20.64 ± 0.39 pg/uL, n = 3, P = 0.007). PLmedia from normoxic and hypoxic placentae reduced sensitivity to ACh (–logEC50, Normoxia: (–)PLmedia: 7.48 ± 0.03 vs. (+)PLmedia: 6.96 ± 0.10, n = 4, P = 0.02; Hypoxia: (–)PLmedia: 7.35 ± 0.35 vs. (+)PLmedia: 6.70 ± 0.29, n = 3, P = 0.08). Conclusion A placental cell model of mitochondrial stress results in cell death and release of mtDNA, while a hypoxic model of stress results in release of mtDNA without cell death. Placental factors decrease resistance artery sensitivity to vasodilators in both normoxic and hypoxic conditions, indicating that the placenta contributes to maternal vascular tone in healthy pregnancies and in pregnancies complicated with reduced perfusion. Ongoing studies investigate the vasoactive potential of placenta-derived cell-free mtDNA.