Inhibition of Mitochondrial Respiratory Chain Complex I Induces Vascular Endothelial Cell Apoptosis and Release of Mitochondrial DNA

Cushen, Spencer
Riaz, Maryam
Phillips, Nicole
Goulopoulou, Styliani
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Purpose: Vascular endothelial oxidative stress is a common feature of preeclampsia, a pregnancy specific hypertensive syndrome with high incidence of maternal and fetal mortality and morbidity. Cellular oxidative stress can lead to cell death, which promotes the release of cellular constituents (e.g. mitochondrial fragments) into the extracellular space. Circulating cell-free mitochondrial DNA (mtDNA) concentrations are increased in pregnant women with preeclampsia. The main objective of this study was to determine the mechanisms by which vascular endothelial cells may contribute to this increase in cell-free mtDNA. The hypothesis was that mitochondrial complex I inhibition results in extrusion of mtDNA from vascular endothelial cells via cell death-dependent mechanisms. Methods: Human umbilical vein endothelial cells (HUVEC, Lonza) were grown to 80-90% confluency before treatment with a mitochondrial complex I inhibitor (Rotenone: 5, 10, 25 μM - 4 h). Immunocytochemistry was used to confirm that HUVEC maintained endothelial cell characteristics. Cell death (apoptotic and non-apoptotic) was quantified using flow cytometry (staining for Annexin V and propidium iodide). mtDNA was measured on total nucleic acid extracts from cell culture supernatants using absolute real-time PCR techniques. Results: Treatment of HUVECs with rotenone increased early apoptosis and late apoptosis/necrosis [5μM (n=7), Veh: 11.16 ± 1.96% vs Rotenone: 14.74 ± 1.96% p=0.0159; 10μM (n=7), Veh: 10.54 ± 1.93% vs Rotenone: 14.83 ± 2.60% p=0.0033; 25μM (n=7), Veh: 10.34 ± 1.85% vs Rotenone: 15.87 ± 3.023% p=0.0002; 1-way ANOVA followed by Sidak’s post-hoc test]. Concentrations of mtDNA in HUVEC supernatant were increased in HUVECs treated with 5 μM of Rotenone [Veh (n=5): 2.45 ± 0.05 pg/mL vs. Rotenone (n=6): 3.65 ± 0.39 pg/mL, p=0.0700; Sidak’s post-hoc test]. Higher concentrations of Rotenone had no effect on concentrations of extracellular mtDNA (p [greater than] 0.84). Conclusions: Mitochondrial oxidative stress due to inhibition of mitochondrial respiratory chain complex I induces vascular endothelial cell death. Extrusion of mtDNA from apoptotic and necrotic endothelial cells may contribute to increased circulating mtDNA concentrations in preeclamptic pregnancies. Future studies will test this hypothesis using integrative pharmacological and physiological approaches.