Measurement of Cellular Energetics Under Different Oxygen Tensions

 

stress and activation of the UPR are increasingly implicated in atherosclerosis and inflammatory pathologies, the mechanisms involved in triggering this response are poorly defined. in this study, we examined the role of the myeloperoxidase (MPO)derived oxidant hypochlorous acid (HOCl) in the induction of ER stress within cells of the vasculature, as this oxidant has been strongly implicated in mediating vascular cell dysfunction and contributing to the development of atherosclerosis. Exposure of endothelial, vascular smooth muscle, and human monocyte-derived macrophage (HMDM) primary cell cultures to HOCl led to a time and dose-dependent increase in the PERKcontrolled effector molecules of the UPR including ATF4, ATF3, and CCAAT/enhancer binding protein homologous protein (CHOP), concurrent with increased mitochondrial dysfunction and cell death. Evidence was also obtained for the increased expression of several key inflammatory mediators including tumour necrosis factor alpKD 71)Į and interleukin 8 (IL8). Conversely, there were no increases in the IRE1-regulated spliced X-box binding protein 1 (sXBP1), the downstream ER degradation-HQKDQFLQJ Į-mannosidase-like protein (EDEM), or ATF6 expression. These findings support HOCl as a potential inducer of ER stress, which may promote the development of atherosclerosis, given the compelling evidence for a role of MPO in this disease.

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 doi: 10.1016/j.freeradbiomed.2013.10.561

   Measurement of Cellular Energetics Under Different Oxygen Tensions Bradford G Hill1 and Timothy D Cummins1 1 University of Louisville, United States

   Role of the Myeloperoxidase-Derived Oxidant Hypochlorous Acid in the Induction of Endoplasmic Reticulum Stress in Cells of the Vascular System Benjamin Rayner1,2, Anna Forsman Quigley1, and Clare Hawkins1,2 1 Heart Research Institute, Sydney, Australia, 2University of Sydney, Australia Endoplasmic reticulum (ER) stress and markers of the unfolded protein response (UPR) signaling cascade are prominent features of both early and advanced atherosclerotic lesions. the UPR involves the activation of three pathways: protein kinase RNA-like ER kinase (PERK), inositol-requiring protein-Į ,5(  and activating transcription factor (ATF) 6, which block protein synthesis, degrade non-essential mRNA, and regulate expression of genes required to maintain cellular homeostasis. Although ER

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The advancement of high-throughput techniques to measure cellular bioenergetics has increased our capacity to understand the mechanisms by which metabolism integrates with cell phenotype and function. Cells are typically cultured and assayed under normoxic conditions (i.e., 21% O 2), which could pose a problem, as cells function under much lower oxygen tensions in vivo. Therefore, to understand how metabolism may contribute to cell phenotype and responses to stress, it is important to have the capacity to model this scenario as closely as possible. in this study, we standardized methodology to measure cellular bioenergetics under different oxygen tensions. an XF24 analyzer (Seahorse Biosciences) was placed inside a SCI-tive-N hypoxia workstation (Ruskinn Technology), and mitochondrial and glycolytic function of cells of cardiac origin were measured. the workflow involved a  K ³GH-JDVVLQJ´ SHULRG in the hypoxia workstation followed by measurement of the oxygen consumption and extracellular acidification rates using the XF analyzer. the oxygen tension recorded by the XF analyzer was corrected under conditions of hypoxia by injecting sodium sulfite into chosen wells of the XF plate. Using a hypoxia software recalculator, correct oxygen consumption rates were obtained for cellular environments with as low as 3% O2. This methodology could be important for understanding how cellular bioenergetics under different oxygen tensions contribute to changes in cell phenotype or responses to stress.

 doi: 10.1016/j.freeradbiomed.2013.10.562

  

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