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The role of mitochondria in defence mechanisms of human endothelial cells

Introduction: Mitochondria are considered to be the powerhouse of the cell being the primary generators of ATP, they also have numerous other important functions including; being the main generator of reactive oxygen species (ROS) and a central role in apoptosis. As the main intracellular source of ROS, many people believe that mitochondria play a significant role in ageing. Senescence is associated with ageing and has been associated with atherosclerotic vascular disease. The concept of human cells lacking functional mitochondria (Rho 0 cells) is not new and was first described by Attardi et al in 1989. However, most of this work has been done on immortalised cell lines. Aims: To see if it is possible to generate and characterise Rho 0 human endothelial cells. To use these cells as a tool to investigate the mechanisms by which they respond to stress and whether differences in ROS production and/or antioxidant defences account for any differences observed. Methods: Human Umbilical Vein Endothelial Cells (HUVEC) were grown in media supplemented with glucose and uridine in the presence of low dose ethidium bromide. Rho 0 status of the cells was confirmed by auxotrophy for uridine, quantitative PCR for mitochondrial-encoded gene expression and western blots for mitochondrial-encoded proteins. Results: The Rho 0 status of the cells was confirmed by; auxotrophy for uridine (Rho 0 cells die in medium lacking uridine), absence of mitochondrial-encoded genes (subunit-1 of complex IV and subunit-6 of subunit V) and lack of expression of the mitochondrial-encoded protein subunit-1 of complex IV. Rho 0 cells are resistant to both stress-induced senescence and apoptosis. They produce less ROS and have upregulated antioxidant defences. Conclusions: It is possible to grow Rho 0 HUVEC. These cells are a useful tool for studying the role of mitochondria in senescence and apoptosis in the cardiovascular system.
Date January 2010
CreatorsWilkinson, J. R.
PublisherUniversity College London (University of London)
Source SetsEthos UK
Detected LanguageEnglish
TypeElectronic Thesis or Dissertation

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