The current study was undertaken to address the role of mitochondrial reactive oxygen species (ROS), and hypoxia inducible factor-1 alpha (HIF-1α) signaling pathway in the protection against high glucose levels in brain endothelial and NT2 neuron-like cells. Rat brain endothelial cells (RBE4) treated with non-toxic concentrations of cyanide (≤1 μM; 1h) exhibited an increase in ROS levels, particularly hydrogen peroxide (H(2)O(2)). Cyanide also induced a modest mitochondrial depolarization, an increase in oxygen consumption and a structural (smaller mitochondria) and spatial (perinuclear region) reorganization of mitochondrial network. The stabilization and nuclear activation of HIF-1α in the presence of cyanide were also observed, which resulted in an increase in vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS) and erythropoietin (EPO) protein levels reflecting an adaptive response. Importantly, preconditioning induced by cyanide protected brain endothelial cells against high glucose-mediated damage by the prevention of apoptotic cell death. In mitochondrial DNA-depleted NT2 (NT2 ρ0) cells, cyanide (0.1 μM) was unable to stimulate ROS production and, consequently, protect against glucotoxicity. Conversely, in NT2 cells, the parental cells with functional mitochondria, cyanide significantly increased ROS levels protecting against high glucose-induced neuronal cell loss and activation of caspase-3. The free radical scavenger N-acetyl-L-cysteine and the specific HIF-1α inhibitor 2-methoxyestradiol completely abolished the protective effects of cyanide preconditioning. Altogether our results demonstrate that mitochondrial preconditioning induced by cyanide triggers a protective response mediated by mitochondrial ROS and HIF-1α activation and signaling, which render brain endothelial and neuronal cells resistant against glucotoxicity.
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Acknowledgements This work is supported by the Fundação para a Ciência e a Tecnologia and Fundo Europeu de Desenvolvimento Regional (PTDC/SAU-NMC/110990/2009) and European Foundation for the Study of Diabetes and Servier 2007. Sónia C. Correia has a PhD fellowship from the Fundação para a Ciência e a Tecnologia (SFRH/BD/40702/2007).
- Caspase 3/metabolism
- Cell Line
- Cells, Cultured
- Endothelial Cells/cytology
- Hydrogen Peroxide/metabolism
- Hypoxia-Inducible Factor 1, alpha Subunit/metabolism
- Membrane Potential, Mitochondrial/drug effects
- Mitochondria/drug effects
- Nitric Oxide Synthase Type III/metabolism
- Oxygen Consumption/drug effects
- Potassium Cyanide/pharmacology
- Reactive Oxygen Species/metabolism
- Vascular Endothelial Growth Factor A/metabolism