Time-Dependent Effect of Advanced Glycation End Products Treatment on Hypoxia Inducible Factor-1  Activation and Partnering ABSTRACT Advanced glycation end products (AGEs) are produced through non- enzymatic glycation reactions between reducing sugars and free amino acids. AGEs have been shown to induce angiogenesis and apoptosis activation through differential partnering of hypoxia inducible factor-1α (HIF-1α) to arylhydrocarbon nuclear translocator (ARNT) and p53. ARNT and p53 have been shown to increase vascular endothelial growth factor (VEGF) and apoptosis, respectively. While relationships among these have been investigated, the mechanism by which activation occurs remains unclear. The goal of this study is to determine whether AGE preferentially activates and couples HIF-1  in a time-dependent fashion. Human retinal pigment epithelial (ARPE) cells were treated with 50 or 100 μg/ml AGEs for 0-24 hours in presence or absence of serum and total levels of HIF-1α and its partnering to ARNT and p53 were determined through western blot analysis. The expected results are that AGEs effect HIF-1  levels in a time-dependent manner and there is preferential partnering to p53 during early exposure and to ARNT during prolonged exposure. If these results hold true, then the significance of this study could assist researchers in finding new venues for the treatment of diabetic retinopathy. INTRODUCTION Diabetes affects 75 million individuals in the United States and throughout the world, resulting in many complications such as, blindness, high blood pressure, compromised immune function, and peripheral vascular disorders. In fact, diabetic retinopathy is the leading cause of blindness in the United States. Studies have shown that this can be a result of proliferation and death in the endothelial and smooth vascular cells of the eye. Unfortunately, the nature of relating diabetes to this proliferation is unclear (Treins et al 2001, Stitt 2001). While many groups are researching the mechanisms by which this occurs, little is known at this time. Diabetics suffer from hyperglycemia, or high circulating levels of glucose. As a result, advanced glycation end products (AGEs) are produced through non-enzymatic glycation reactions. AGEs accumulate throughout the body in diabetics, including in the eye, have been shown to correlate with the progression of diabetic retinopathy. They have also been shown to cause proliferation and apoptosis in cells, which suggests that AGEs play an important role in diabetic retinopathy disease state (Stitt 2001). Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that is always present in the system, and induced under low oxygen conditions. Under normoxia, approximately 20% oxygen, it is rapidly ubiquitinated and then degraded. Recent data has shown that with an increase in AGEs both HIF-1α protein and mRNA levels increase as well. In turn, HIF-1α greatly accumulates and can bind with HIF-1β, which is also known as arylhydrocarbon nuclear translocator (ARNT). This binding forms the HIF-1 complex and has been shown to increase the expression of vascular endothelial growth factor (VEGF) through binding on the hypoxia response element (HRE) (Figure 1) (Treins et al 2001). An increase in VEGF expression has been shown to lead to blood vessel proliferation, or angiogenesis (Goodsell 2003). Under hypoxic conditions, less than 5% oxygen, HIF-1α partners with p53, a tumor suppressor gene resulting in increase of apoptosis, or cell death (Gardner et al 2001). It is thought that preferential partnering of HIF-1α with either ARNT or p53 is mediated on its phosphorylation status; ARNT with binding hyper-phosphorylated and p53 interaction with the hypo-phosphorylated HIF-1  (Treins et al 2001, Gardner et al 2001). While HIF-1  activation may be important in mediating diabetic retinopathy, the role of AGEs in this process is yet to be elucidated. OBJECTIVES 1.To determine whether AGE treatment has a time-dependent effect on total HIF-1  protein levels. 2.To determine whether preferential partnering of HIF-1  to ARNT or p53 is time-dependent. METHODS Cell Culture and Treatment Human retinal pigment epithelial (RPE) cells were cultured and treated in DMEM/F12+10% fetal bovine serum. Cells were treated with 100 or 500 μg/ml human glycated albumin AGEs (Sigma), 200 μM CoCl 2 (positive control), or no treatment for 0-24 hr. Protein Isolation and Quantification Cells were washed with ice cold PBS. RIPA buffer (350 μl) with Protease inhibitors (Sigma). Samples were placed on ice for 10 min. Cells were scraped and put into tubes. Samples were vortexed for 15 min at 4°C. Samples were cleared at 14k for 5 min at 4°C. Proteins quantified using BCA assay (Pierce) according to manufacturer’s instructions. Immunoprecipitation 500 μg total protein, 2.5 μl IgG, 20 μl Protein G-Agarose, and 1 μl Anti-HIF-1  Antibody (Novus) incubated overnight at 4°C. Samples were centrifuged at 15k for 30 sec at 4°C. Pellets were taken up in 20 μl Lamelli Buffer (SDS-PAGE) and boiled for 5 min. Western Blot 7.5% SDS-PAGE gel was loaded with 12 μl IP sample. Run at 170 V for 1 hr. Transferred onto a PVDF membrane at 100 V for 1 hr. PVDF membranes were incubated overnight with a primary antibody: 1:400 Anti-HIF-1  antibody (Novus) 1:1000 Anti-p53 antibody (Novus) 1:1500 Anti-ARNT antibody (Novus) A secondary antibody, 1:4000 Anti-mouse alkaline phosphatase antibody was applied for 30 min. Membranes were developed with BCIP/NBT for 10 min at room temperature. PREDICTED RESULTS RESULTS HIF-1  was immunoprecipitated at 2 and 4 hr txt (Figure 2). However, because the positive control, CoCl 2 did not show up, we have predicted results. Predict that, HIF-1  will increase in a time-dependent manner (Figure 3). Predict that, HIF-1  will partner with p53 during temporary exposure, up to 6 hr (Figure 4). Predict that, HIF-1  will partner with ARNT, as seen by VEGF protein levels during prolonged exposure, > 4 hr (Figure 4). FUTURE DIRECTIONS Increase sensitivity of assay. Use enhanced horseradish peroxidase- coupled secondary antibody with chemiluminescence (ECL) reagent for detection, increasing sensitivity. Use an alternative anti-HIF-1  antibody with increased sensitivity. Investigate the effects of serum vs. serum-starved cell cultures at various times prior and during treatment. Investigate phosphorylation status of HIF-1  as it correlates with the preferential partner in response to AGE treatment. Change cell culture model; smooth muscle or endothelial cells. LITERATURE CITED Diabetic retinopathy images. Available from: Accessed 2003 March 24. Gardner, L.B., Li, Q., Park, M.S., Flanagan, W.M., Semenza, G.L., and Dang, C.V The Journal of Biological Chemistry 276(11): Goodsell, D.S Stem Cells 21: Lu, M., Kuroki, M., Amano, S., Tolentino, M., Keough, K., Kim, I., Bucala, R., and Adamis, A.P Journal of Clinical Investigations 101(6): Stitt, A.W British Journal of Opthamology 85: Treins, C., Giorgetti-Peraldi, S., Murdaca, J., and Van Obberghen, E The Journal of Biological Chemistry 276(47): Yamagishi, S., Inagaki, Y., Okamoto, T., Amano, S., Koga, K., Takeuchi, M., and Makita, Z The Journal of Biological Chemistry 277(23): ACKNOWLEDGEMENTS Thank you to Dr. Kaltreider for constant guidance, support, and encouragement. Julie Kopp Department of Biological Sciences, York College of Pennsylvania RESULTS HIF-1  Figure 3. Predicted results of western blot analysis of IP samples. RPE cells treated with 100 μ g/ml AGEs probed with Anti-HIF-1  antibody over time (hr), 200 μ M CoCl 2 for 4 hr, and no treatment (Cont). AGE TXT (hr) CoCl Cont p53 VEGF Figure 4. Predicted results of western blot analysis of IP samples. RPE cells treated with 100 μ g/ml AGEs probed with Anti-p53 antibody or anti-VEGF antibody over time (hr), 200 μ M CoCl 2 for 4 hr, and no treatment (Cont). AGE TXT (hr) Diabetic retinopathy (eyesearch.com). CONCLUSIONS HIF-1  could only be seen at 2 and 4 hr, leading us to believe that this txt induces the highest level of HIF-1 . However, due to technical problems and low sensitivity this result needs to be repeated because the positive control did not work. Based on our predicted results, HIF-1α induction will show a time-dependent increase in response to AGE txt. Based on our predicted results, HIF-1α will preferentially partner with p53 early leading to increased apoptosis. Based on our predicted results, HIF-1α will preferentially partner with ARNT leading to an increased VEGF production during later exposure to AGEs (Lu et al 1998, Yamagishi et al 2002). AGEs HIF-1  p53 Apoptosis ARNT VEGF Hypophosphorylation Short-term Exposure Angiogenesis Figure 1. Proposed mechanism. The effect on HIF1  levels and its preferential partnering upon an increase of AGE levels over time leading to disease (Adapted from Gardner et al 2001, Treins et al 2001). Hyperphosphorylation Prolonged Exposure CoCl 2 Figure 2. Western blot analysis of IP samples. RPE cells were treated with 100 μ g/ml AGEs over time (hr), 200 μ M CoCl 2 for 4 hr, or no treatment (Cont) and probed with Anti-HIF-1α antibody. AGE TXT (hr) HIF-1  IgG mw kDa Cont CoCl Cont