1. Dose Dependant Effect of Advanced Glycation End Products on Human ARPE-19 Cell Viability Duane C. Kline, Department of Biology York College of Pennsylvania Abstract Diabetic retinopathy, which results in thickening and death of the blood vessels, is a disease that has been linked to high concentrations of circulating advanced glycation end products (AGE). The mechanism of AGE in this condition is not fully understood, but recent work has shown possible similar pathogenic events that are caused by Hypoxia Inducible Factor (HIF-1α) activation resulting in angiogenesis and apoptosis. The goal of our study was to determine the effect AGE concentration resulted in a change of human retinal cell viability. We treated retinal cells in media with 0 to 500 µg/ml of AGE in serum free media with or without 0.2% BSA, were treated for 24 hr. Cell viability was assessed using a standard CFDA assay. We observed no difference between serum free and 0.2% BSA groups. There was significant difference (p< 0.01) between non-treatment group with the 50 µg/ml and 500 µg/ml groups in 0.2% BSA. We observed a bi-phasic event with two groups of cell death occurring in the 25 µg/ml to 50 µg/ml and post 100 µg/ml AGE dosages. The data suggests a possible role of AGE dosages in the development of altered retinal cell viability suggesting a role in diabetic retinopathy. Introduction Diabetes is a growing epidemic worldwide and across the United States (1,2). The World Health Organization census figures in 1985 found 30 million reported cases of diabetes. Currently it is estimated that 177 million cases worldwide and the prediction for 2025 has the number of cases reaching 300 million (1). Diabetes is the 7 th leading killer in the United States and increases the incidence rates of other illnesses including retinopathy, neuropathy, renal failure and heart disease (2). Diabetes results from inefficient utilization of insulin that results in high levels of glucose circulating throughout the body, termed hyperglycemia. These conditions result in an increase of glycation of bio-chemicals through non enzymatic reaction with free sugars, DNA, lipids, and amino acids, creating advanced glycation end products (3). AGEs have been recently linked to diabetic retinopathy as one of the major contributors to the disease (4). Retinopathy is an illness that causes damage to the small blood vessels located inside the eye (Fig. 3). AGEs have been shown to localize in the blood vessels, vascular basement membranes, and retinal pericytes within diabetic patients (3). The blood vessels undergo stages of thickening, loss of vascular permeability, death, and re-growth that eventually lead to blindness. When non-diabetic rats intravenously injected with exogenous AGEs their eyes undergo similar pathogenesis of diabetic retinopathy, which resulted in a loss of pericytes (4). AGEs and hypoxic conditions have been documented to activate HIF-1α, a translation factor that regulates genes involved in apoptotic or angiogentic responses(5,6,7). Through the induction of VEGF (vascular endothelial growth factor) and p53 (tumor suppressor protein), respectively. Figure 1 shows the low AGE concentration pathway activating HIF-1α binds ARNT (aryl hydrocarbon receptor nuclear translocator) causing angiogenesis while, HIF-1α binding to p53 tumor suppressor protein that initiates an apoptotic cascade at high concentrations (5,7,8,9). While many aspects of this pathway has been observed and characterized under hypoxia conditions, little is know of the molecular effects of AGEs on this pathway. Signaling of the correct pathway is due to hypo- or hyper- phosphorlation of HIF-1α. This concentration can be dependent of available oxygen or the amount of AGE concentration. The goal of our study was to determine the effect of increasing dose of AGE on the viability of retinal cells. Based on diabetic retinopathy pathophysiology we predict that increased AGE dosage will decrease cell viability. From this research, a concentration set point can be obtained to further evaluate AGE interactions and explicate the mechanism of action of the retinal eye cells. Materials and Methods Cell Culture Human ARPE-19 cells (ATCC) were maintained in a Dulbecco’s modified Eagle medium /F12+10% fetal bovine serum (Gibco, BRL) at 37  C at 5% CO 2. Cells were split 1:750 from a T-75, one day prior to treatment into 96-well plate (VWR). 18 hr prior to treatment, cells were shifted to 100 µl incomplete media +/- 0.2% BSA. Chemical Treatments Chemicals were added to cells in 100 µl of incomplete media +/- 0.2% BSA. Cells were treated with a 100 µl dosage of AGE (25, 50,100, 250, and 500 µg/ml) or taxol (100 nM) as a positive control (n=4), then incubated for overnight. Cell Viability Assay After treatment, the cells were washed in 100 µl of PBS. 50 µl of the CFDA (25 mM) solution was added to cells and incubated at 37  C for 2 hr under complete darkness. Viability was measured in the form of cell fluorescence, which was assessed using a Wallac Victor 2 plate reader using 496 nm excitation and 530 nm emission. The fluorescent values were then converted to percent control of treatments verses untreated values. The following statistical analyses were performed: 1-way ANOVA with a Kruskal-Wallis post-test, Mann- Whitney test, and T-test. Figure 3. Progression of Diabetic Retinopathy (Left) A normal eye with retina and blood vessels clearly depicted. (Center) shows an eye with increased number and size of blood vessels. (Right) Eye that has hemorrhaged causing blood to encase the eye (10). Results  No significant difference between AGE treatments in serum free media +/- 0.2% BSA.  AGE concentration of 25 µg/ml in serum free and 250 µg/ml in 0.2% BSA were significantly lower than non-treatment control groups (p<0.05). AGE concentrations of 50 µg/ml and 500 µg/ml in 0.2% BSA were significantly lower than non-treatment control groups (p<0.01).  Taxol treatments caused a significant reduction in viability from non-treatment control cells. (p<0.05). Discussion We observed a bi-phasic trend in AGE treated +/- 0.2% BSA on cellular viability. There was an initial decrease in viability (25-50 μg/ml), with a recovery to non- treatment control level viability (100 μg/ml), followed by a gradual decrease in viability at increasing AGE concentrations (>100 μg/ml). Explanation of this trend maybe best understood using elements known to diabetic retinopathy pathophysiology. Diabetic retinopathy is characterized by initial cellular death, which we may be observing in this study at lower doses. This could increase the amount of the p53 tumor proteins resulting in apoptosis. The increase of cellular loss triggers a proliferatory response, which would explain the recovery stage and possible activation of VEGF. In the body, thickening of the basement layers have been recorded in both human patients and rat models (3,11). We found that after 100 μg/ml the viability decreased, suggesting a second activation of p53 or unspecific mechanisms due to increased AGE concentrations. Due to limitations, we were not able to fully elucidate the role of AGE in diabetic retinopathy in this study. In future studies, we hope to use the following tests to aid in modification of our current model of AGE manifestation in retinal cells: Caspase-3 Activity to measures apoptosis VEGF Expression to demonstrate angiogenesis We predict increased Caspase-3 activity in the 25-50 μg/ml and 250-500 μg/ml, because of Caspase-3 activity is a commitment step in the apoptotic cascade. The Caspase-3 activity would be very low at the 50-100 μg/ml treatments, while VEGF expression would be increased due to cells undergoing proliferation. The future of AGE research could change the methods for diagnosing AGE induced diabetic retinopathy. Cellular samples could be taken from patients to see the amount of AGE stress that has endured. Currently, AGEs are being used to stiffen and strengthen tissues cultured for transplants. Researchers have exploited the AGE properties by allowing tissues to be incubated in a glucose rich cell media and allowed to grow for up to 10 weeks with positive results (12). AGE research will be very beneficial to the future patients with a vast number of novel technologies that can be created. Literature Cited 1.World Health Organization. September 2002. The cost of diabetes; Fact sheet 236. Available from: http://www.who.int/mediacentre/factsheets/fs236/en/print.html. Accessed on 21 October 2003. 2.Freid VM, Prager K, MacKay AP, Xia H. Chartbook on Trends in the Health of Americans. Health, United States, 2003. Hyattsville, Maryland:National Center for Health Statistics. 2003. Accessed from: http://www.cdc.gov/nchs/hus.htm. Accessed on 21 October 2003. http://www.cdc.gov/nchs/hus.htm 3.Stitt A. 2001. Advanced glycation: an important pathological event in diabetic and age related ocular disease. The British Journal of Ophthalmology. 85:746-753. 4.Xu, X., et al. 2003. Exogenous advanced glycoslation end products induce diabetes-like vascular dysfunction in normal rats: a factor in diabetic retinopathy. Graefe’s Arch. Clinical Exp. Ophthalmology. 241:56-62. 5.Yamagishi, S. et al. 2002. Advanced glycation end product-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: (23)20309-20315. 6.Jewell, U. et al. 2001. Induction of HIF-1a in response to hypoxia is instantaneous. The FASEB Journal. 15:1312-1314. 7.Jiang, B. et al, 1996. Dimerization, DNA binding and trans-activation properties of hypoxia-inducible factor 1. The Journal of Biological Chemistry. 227: (30)17771-17778. 8.Chen et al., 2003. Direct interactions between HIF-1α and Mdm2 modulate p53 function. The Journal of Biological Chemistry. 278: (16) 13595-13598. 9.Treins et al. 2001. Regulation of vascular endothelial growth factor expression by advanced glycation end products. The Journal of Biological Chemistry. 276:43836-43841. 10.New England Eye Center. 2000. Available from: http://www.neec.com/Vitreoretinal_Disease_Diabetic_Retinopathy.html. Date accessed 2003 September 8. http://www.neec.com/Vitreoretinal_Disease_Diabetic_Retinopathy.html 11.Tsilibary, E. 2003. Microvascular basement membranes in diabetes mellitus. Journal of Pathology. 200:537-46. 12.Girton, T. et al.1999. Exploiting glycation to stiffen and strengthen tissue equivalents for tissue engineering. Journal of Biomedical Materials Research. 46: 87–92. Figure 1. Proposed AGE pathway in retinal cells. At low concentrations of AGE, HIF-1α (Hypoxia Inducible Factor) binds ARNT (aryl hydrocarbon receptor nuclear translocator) inducing VEGF (vascular endothelial growth factor) which causes angiogenesis. At high AGE concentration, HIF-1α binds to initiate p53, which causes apoptosis (5,7,8,9). Acknowledgements I would like to thank the following professors for their assistance during my thesis research: Ron Kaltreider, Ph.D., Mentor Karl Kleiner, Ph. D.