1. Dose Dependent Effect of Advanced Glycation End Products on Human Retinal Pigment Epithelial cell viability Jennifer Winemiller, Department of Biological Sciences, York College of Pennsylvania Abstract Diabetes is caused by high levels of glucose circulating through the body, also known as hyperglycemia. It has been shown that accumulation of Advanced Glycation End Products (AGEs) in the retina can lead to a loss of renal function. The goal of this study is to determine the effect of AGEs on Retinal Pigment Epithelial (RPE) cell viability through the use of different AGE doses at 24 and 48 hours, and one week. A CFDA-AM test was used to measure cell viability. At 24 and 48 hours, cell viability was reduced by about 50% at 100ug/ml. During the one week trial, approximately 50% reduced viability is noted at 0.001ug/ml. Throughout the study, all three experiments showed a decrease in viability over time showing that time had an effect on viability as well as AGE dose concentration which seems to support my hypothesis. Introduction Diabetes is caused by high levels of glucose circulating through the body, also known as hyperglycemia. With these high levels, glucose can react with amino acids to produce early glycation products. From there, these products can undergo more complex reactions to irreversibly become Advanced Glycation End Products (AGEs) (Yamagishi 2002). It has been shown that accumulation of AGEs can lead to a loss of renal function (Yamagishi 2002). As this loss advances, diabetic retinopathy, which is the thickening and death of blood vessels, eventually results in blindness. One of the believed causes of diabetic retinopathy is the release of angiogenic factors such as Vascular Endothelial Growth Factor (VEGF) (Treins 2001). There has been evidence that VEGF is controlled by hypoxia-inducible factor 1 (HIF 1), especially under hypoxic conditions (Wartenberg 2001). HIF 1 is a transcription factor made up of HIF 1α and HIF 1β, also known as aryl hydrocarbon nuclear translocator protein or ARNT. Under hypoxic conditions, HIF 1α will bind with either ARNT or p53, a tumor suppresser gene. If there are high levels of phosphorylation, HIF 1α reacts with ARNT resulting in higher levels of VEGF, which ultimately leads to angiogenesis. However, if there are lower levels of phosphorylation, HIF 1α reacts with p53, resulting in apoptosis. The dose at which this switch occurs has yet to be determined. My hypothesis is that cell viability will decrease in a dose and time dependent fashion. Methods 24 hour treatments  RPE-19 cells were divided in a 1:750 split into serum and serum free wells on a 96 well plate. Cells were given AGE doses of 25, 50, 100, 250, and 500 µg/ml with each group containing 3 trials. After 24 hours they were treated with 50 µl CFDA-AM solution (Molecular Probe) and read in the Wallac Plate Reader to test cell viability. 48 hour treatment  At 48 hours RPE cells were divided in a 1:750 split into serum and serum free wells on a 96 well plate. Cells were given AGE doses of 25, 50, 100, 250, and 500 µg/ml with each group having an containing 3 trials. After 48 hours they were treated with µl CFDA-AM solution and read in the Wallac Plate Reader to test cell viability. One week treatment  RPE cells were divided in a 1:30 split in serum free wells on a 6 well plate. Cells received AGE doses of 0.001, 0.01, 0.1, 1,10, and 100 µg/ml. Cell viability was then measured after 7 days with 50 µl CFDA-AM solution and read in the Wallac Plate Reader to test cell viability. Data Analysis  The values used in graphing were obtained by taking the average of the absorbances measured by the Wallac plate reader and subtracting the background absorbance. Those values were then divided by the percent control to produce an average percent viability with the control representing 100%. Image courtesy of the National Eye Institute Figure 1. The percent viability from the CFDA-AM test of serum free, and serum treated human RPE-19 cells after being treated with different concentrations of AGEs for 24 hours. N= 3± SEM Figure 2. The percent viability from the CFDA-AM test of serum free and serum treated human RPE-19 cells after being treated with different concentrations of AGEs for 48 hours. Also, the percent viability of cells that survived a taxol treatment that acted as a negative control. N= 3± SEM Figure 3. The percent viability from the CFDA-AM test of serum free human ARPE-19 cells after being treated with different concentrations of AGEs for seven days. Results At 24 hours, the serum free and serum cells had the lowest viability at 100 µg/ml. Figure 1. At 48 hours, the serum free cells had the lowest viability at 1000µg/ml AGE. The serum cells had the lowest viability at 250µg/ml. Figure 2. The 7 day treatment showed the lowest viability to be at 0.001 µg/ml. Figure 3. Discussion The 24 and 48 hour treatments both showed decreases in cell viability of approximately 50% at 100 µg/ml. In another study, it was found that cell viability also decreased drastically when treated with AGEs (Chibber 1997). During the one week trial, the viability still decreased by approximately 50%, but at a much lower dose. Only 0.001 µg/ml of AGEs were needed to obtain the reduction in cell viability. Throughout the study, all three experiments showed a decrease in viability over time showing that AGE treatments have both a dose and time dependent effect on viability. Using this data, we can conclude that AGEs, even at a low dose over an extended period of time, can cause decreases in cell viability which will eventually lead to diabetic retinopathy in patients suffering from diabetes. 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Tumor-induced Angiogenesis Studied in Confrontation Cultures of Multicellular Tumor Spheroids and Embryoid Bodies Grown from Pluripotent Embryonic Stem Cells. The FASEB Journal 15: 995-1005. Yamagishi, S., Inagaki, Y., Okamoto, T., Amano, S., Koga, K., Takeuchi, M., and Makita, Z.. 2002. Advanced Glycation End Products-Induced Apoptosis and Overexpression of Vascular Endothelial Growth Factor and Monocyte Chemoattractant Protein-1 in Human Cultured Mesangial Cells. The Journal of Biological Chemistry 277: 20309-20315. Acknowledgements I would like to thank Dr. Ron Kaltreider for all his time and knowledge throughout this process.