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.