1. INTRODUCTION Both Type I and Type II diabetes are characterized by increased circulating levels of glucose (hyperglycemia). A major complication of chronic hyperglycemia is retinopathy, a common cause of blindness in diabetes patients. Hyperglycemia has been shown to induce the production of advanced glycation end products (AGEs) through non-enzymatic reactions between glucose and blood macromolecules (Wautier et al. 2001). AGEs have been shown to both stimulate vascular endothelial growth factor (VEGF) and induce apoptosis (Treins et al. 2001). VEGF is a potent stimulator of blood vessel proliferation and has been linked to morphological and molecular changes within retinal epithelial and endothelial cells in the occurrence of diabetic retinopathy. VEGF is regulated by the transcription factor, hypoxia inducible factor (HIF)-1 (Semenza 2000). HIF-1 is composed of two subunits, HIF-1α and HIF-1β (also known as the aryl hydrocarbon nuclear translocator; ARNT). Whereas ARNT is constitutively expressed in the cell nucleus, HIF-1α is rapidly induced by changes in oxygen concentrations. During normoxic conditions (20% O 2 ), HIF-1α is degraded through a ubiquitin- mediated process. However, under hypoxic conditions (< 5% O 2 ), HIF-1α binds with either ARNT or p53, a tumor suppressor gene. Binding to ARNT results in increased VEGF transcription and expression through increased binding to and transactivation of hypoxia response element (HRE) (Suzuki et al. 2001). Binding to p53 results in increased apoptosis and degradation of the HIF-1α protein. While AGEs have been shown to increase VEGF expression and induce apoptosis, little is known about their affect on HIF-1α protein levels. Therefore, the goal of this study is to determine the affect of increasing doses of AGE on HIF-1α protein. OBJECTIVE: ● Determine HIF-1α protein levels in response to increasing concentrations of AGE in cultured human cells. HYPOTHESIS: ● AGE exposure increases HIF-1α protein levels in a dose-dependent fashion. ABSTRACT A major complication of chronic hyperglycemia is diabetic retinopathy, a leading cause of blindness in the U.S. Hyperglycemia has been shown to induce the production of advanced glycation end products (AGEs). AGEs activate vascular endothelial growth factor (VEGF), which stimulates blood vessel proliferation and has been linked to changes within the retinal epithelial cells in diabetics presenting retinopathy. VEGF is regulated by the transcription factor, hypoxia inducible factor (HIF)-1, which is composed of two subunits, HIF-1α and the aryl hydrocarbon nuclear translocator (ARNT). HIF-1α partners with either ARNT or p53. Our study examines the role of AGE treatment on HIF-1α activity. Human Retinal Pigment Epithelial cells were treated for 4 hr with increasing concentrations (0-500 μg/ml) AGE, and total HIF-1α levels were determined through western blot. While AGEs alter HIF-1α protein levels, the results do not suggest a dose-dependent relationship. The Role of Advanced Glycation End Products (AGEs) on HIF-1α Protein Levels Susan Curilla and Ronald C. Kaltreider Department of Biological Sciences, York College of Pennsylvannia Figure 1. Proposed AGE-induced expression of HIF-1α and partnering with ARNT and p53. (Adapted from Michael Ihnat, PhD.) METHODS Cell Culture Human ARPE-19 cells (Arising Retinal Pigment Epithelial cell line; ATTC no. CRL-2302) were maintained in 75 cm 2 flasks (Costar) containing 10% fetal bovine serum at 37 o C with 5% CO 2.  Chemical Treatments Cells were treated with increasing concentrations (0–500 μg/ml) of AGE (human glycated albumin; Sigma) for 4 hr. Another set of cells were serum-starved for 24 hr prior to chemical treatments. Serum-starved cells often show a greater induction of the HIF- 1α protein. Positive controls consisted of 1) RPE cells treated with 200 μM cobalt chloride (CoCl 2 ) (4 hr), which induces HIF-1α, and 2) RPE cells maintained in 5% O 2 for 48 hr to stimulate hypoxia.  Protein Isolation 350 μL ice-cold RIPA buffer with protease inhibitors (Complete™ tablets; Roche) was added to each flask. Flasks were shaken gently at 4 o C for 10 min, scraped, and centrifuged at 12K for 3 min to clear debris. Protein concentrations were determined using the BCA method (Pierce).  Western Blot Analysis Samples were electrophoresed on a 7.5% SDS-polyacrylamide gel (120 V) for 1 hr, and then transferred to a PVDF membrane (100 V for 1 hr). HIF-1α protein levels were determined using a HIF-1α antibody (1:400; Novus) and secondary anti-mouse HRP (1:4000; Pierce). Bands were visualized using ECL (Pierce) as per the manufacturer’s specifications. Band intensity was quantified using Scion Image software. Figure 2. Effect of AGE on total HIF-1α protein levels in RPE cells maintained in the presence of serum. (A) Western blot of HIF-1α protein from cells treated with increasing concentrations of AGE (0-500 μg/ml). Positive controls were cells treated with 200 μM CoCl 2 and 5% O 2. (B) Graphical representation of HIF-1α levels as a percent of untreated control cells. The dashed line represents the HIF- 1α protein level in untreated cells. Figure 3. Effect of AGE on total HIF-1α protein levels in serum-starved RPE cells. (A) Western blot of HIF-1α protein from cells treated with increasing concentrations of AGE (0-500 μg/ml). Positive controls were cells treated with 200 μM CoCl 2 and 5% O 2. (B) Graphical representation of HIF-1α levels as a percent of untreated control cells. The dashed line represents the HIF-1α protein level in untreated cells. RESULTS RPE Cells Maintained in Serum Untreated cells showed little or no expression of HIF-1α (Figure 2A). Total HIF-1α protein levels increased approximately 2-fold in all AGE treatments (50-500 μg/ml) compared to the control (Figure 2B). Maximal induction of HIF-1α occurred at 100 μg/ml AGE (Figure 2B). Total HIF-1α protein levels in AGE treatments and the 5% O 2 positive control were similar (Figure 2B). Serum-Starved RPE Cells Total HIF-1α protein levels were higher in the untreated serum- starved cells than in the untreated cells maintained in serum (Figure 2A vs. 3A) Maximal induction of HIF-1α occurred at 500 μg/ml AGE which was a 1.5-fold increase compared to the control (Figure 3B). CONCLUSIONS Total HIF-1α protein levels in the cells maintained in serum increased approximately 2-fold in all AGE treatments (50-500 μg/ml) compared to the control. However, these results do not suggest a dose-dependent relationship between AGE exposure and HIF-1α protein levels. Total HIF-1α protein levels in serum-starved cells are not consistent with those of cells maintained in serum; serum- starvation induced expression of HIF-1α in the control. Thus, while a greater abundance of HIF-1α was observed, less induction compared to the control occurred. However, treatment of cells with 250-500 μg/ml AGE stimulated expression of HIF-1α to a level higher than the control. FUTURE RESEARCH Determine HIF-1α protein levels in response to different concentrations of serum. Characterize HIF-1α partnering to ARNT and p53 in response to AGE treatments. LITERATURE CITED Semenza, G. L. 2000. HIF-1: using two hands to flip the angiogenic switch. Cancer Metastasis Review 19: 59-65. Stitt, A. W. 2001. Advanced glycation: an important pathological event in diabetic and age related ocular disease. British Journal of Ophthalmology 85: 746-53. Suzuki, H., Tomida, A.., and Tsuruo, T. 2001. Dephosphorylated hypoxia-inducible factor 1alpha as a mediator of p53-dependent apoptosis during hypoxia. Oncogene 20: 5779-88. Treins, C., Giorgetti-Peraldi, S., Murdaca, J., and Van Obberghen, E. 2001. Regulation of vascular endothelial growth factor expression by advanced glycation end products. Journal of Biological Chemistry 276: 43836-41. Wautier, J. L. and Guillausseau, P. J. 2001. Advanced glycation end products, their receptors and diabetic angiopathy. Diabetes & Metabolism 27: 535-42. www.med.unibs.it/~airc/ sandra/pathology.html. ACKNOWLEDGEMENTS Pennsylvannia Academy of Science for Grant Support Michael Ihnat, PhD. for his technical assistance and support www.med.unibs.it/~airc/ sandra/pathology.html (Stitt 2001)