Effect of oscillating glucose and glycated albumin on reactive oxygen species and angiogenic signaling in ARPE–19 cells

Abstract

Abstract: : Purpose: Cellular response to oscillating glucose levels and glycated albumin in human ARPE–19 cells and in chicken embryo CAMs were investigated. Methods: ARPE–19 cells in culture and CAMs exposed to glycated albumin, anti–oxidants, and oscillating glucose. Digital microscopic images and densitometry of CAMs, western blotting of hypoxia inducible factor 1–alpha (HIF) and superoxide dismutase (SOD–1), and ELISA of VEGF and nitrotyrosine (NT) were done. Results: Glycated albumin (250 and 500 mcg/ml) was found to increase vessel density in the CAM assay and this effect was attenuated by the addition of various anti–oxidants (d–alpha–tocopherol, N–acetylcysteine, MnTBAP). Short–term (8 hr) and long–term (1 wk) exposure of ARPE–19 cells to glycated albumin increased expression of HIF–1alpha, a marker of angiogenesis and altered glucose, as well as a target protein for HIF, the seminal angiogenesis factor VEGF. In addition to HIF and VEGF, long–term exposure to a lower dose of glycated albumin (100 mcg/ml) also increased levels of the oxidative stress marker NT in the ARPE–19 cells. Long–term exposure to glucose oscillating to mimic meals in a diabetic ( a diabetic level of 10 mM glucose alternating with a post–prandial level of 15 mM glucose every 1.5 hr three times daily) also led to increased HIF and VEGF expression with only a small effect on SOD–1 and NT levels. Finally, long–term exposure to oscillating glucose and glycated albumin together resulted in a more pronounced increase in HIF, VEGF, and SOD–1 expression with little difference in NT levels as compared to glycated albumin or oscillating glucose exposure alone. Conclusions:Glycated proteins and oscillating hyperglycemia, early metabolic changes associated with diabetes, caused an increase in angiogenic signaling in ARPE–19 cells and blood vessel formation in the CAM assay, putatively through the generation of ROS. This suggests that HIF could be playing an earlier role in the pathogenesis of proliferative diabetic retinopathy than previously thought.

Keywords: diabetic retinopathy • glycoconjugates/glycoproteins • retinal pigment epithelium