Abstract
Purpose:
Age-related macular degeneration is one of the leading causes of blindness in the world. Recently, a large clinical trial reported that the long-term use of aspirin was responsible for its occurrence.. Interestingly, it has been proved earlier that the effect of aspirin on the mechanisms leading to age-related macular degeneration is different from that of prevention of heart disease. In this study we propose to determine the effects of aspirin and its toxic metabolites on Peripheral Blood Mononuclear Cells(PBMC) and human retinal pigment epithelial cells maintained in culture medium. The initial phase of this study will focus on the apoptotic effects of Aspirin and its metabolites on peripheral blood mononuclear cells with irradiation of blue light, which has been shown to be an inducer of oxidative stress.
Methods:
: Under an IRB approved protocol blood was collected from healthy volunteers who were taking aspirin and others who were not taking aspirin.. Following 1:1 dilution in PBS, 20 mL of whole blood was overlayed onto 10mL of Ficoll Lymphocyte Separation Medium and spun at 1600 RPM for 30 minutes. Peripheral Blood Mononuclear Cells (PBMC) were recovered, pooled, washed, and resuspened in RPMI-1640 growth media with 10% FBS . Pooled cells in the amount of 2x106cells from respective donors were then taken and were challenged with Aspirin, Hippuric Acid, its metabolite at concentrations of 0.325mg/dl, 0.65mg/dl, and 0.975mg/dl. Samples were subjected to overnight blue light (~430nm) . These samples were tested for apoptosis by Annexin V, and Propidium Iodide using flow cytometry.
Results:
In the normal person, when the normal blood is subjected to blue light, there was a 26.77% increase in apoptosis compared to the control. There was a concentration dependent response due to aspirin on apoptosis. In the aspirinated blood when it was subjected to blue light, there was a 22.16% increase in apoptosis compared to the control. A similar augmentation was observed with the addition of blue light.
Conclusions:
These results suggest that blue light and aspirin by themselves cause apoptosis. However, aspirin in addition to blue light causes augmentation in apoptotic rate. Apoptosis is a key factor involved in age related macular degeneration.
Keywords: 426 apoptosis/cell death •
412 age-related macular degeneration •
634 oxidation/oxidative or free radical damage