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L.A. Wiley, J.J. Steinle; Hydrogen Peroxide Induces Apoptotic Markers in Human Retinal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):145.
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To determine the ability of reactive oxygen species (ROS) to induce apoptosis in human primary microvascular retinal endothelial cells (HMRECs).
HMRECs were maintained in media containing a normal, physiological concentration of glucose (5 mmol), supplemented with 20% FBS. For experiments, cells were switched to media containing 5% FBS overnight to avoid apoptosis due to severe serum starvation, as it is known to induce cell death. Cells between passages 2–7 were treated with 1 mmol hydrogen peroxide (H2O2) at varying timepoints in either normal or high glucose (20 mmol). Cells were harvested by scraping for both RNA and protein. Pro– and anti–apoptotic proteins were analyzed by immunoblotting.
H2O2 increased protein expression of pro–apoptotic bax in cells treated in high glucose as early as three hours, compared to cells treated in the presence of normal glucose. Basal levels of bax were also increased after exposure of cells to high glucose alone, compared to normal glucose–treated cells. Hydrogen peroxide treatment produced a concurrent decrease in the anti–apoptotic protein Bcl–2. There was no change in basal levels of Bcl–2 in high versus normal glucose–treated cells. Additionally, the DNA damage response protein, p53, was also upregulated by H2O2 treatment. Treatment with high glucose alone caused no change in basal p53 levels.
Oxidative stress is hypothesized to be one of many factors contributing to the development of retinopathy. It is defined as an increase in the production of ROS, with hydrogen peroxide being one of them. Increased ROS can result from a number of factors, including glucose oxidation and AGE formation, both of which are upregulated in a hyperglycemic environment. Oxidative stress is thought to cause endothelial cell apoptosis, leading to vaso–obliteration, vascular non–perfusion, and ischemia. However, most experiments are conducted using either HUVECs, which are macrovascular or BRECs that are not human. The results above demonstrate the importance of high ROS and high glucose on cultured primary human microvascular retinal endothelial cells. Further understanding of the mechanisms behind oxidative stress induced apoptosis could lead to new therapeutic intervention for diabetic retinopathy.
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