Pharmacological Inhibition of N-Methyl D-Aspartate Receptor Promotes Secretion of Vascular Endothelial Growth Factor in Müller Cells: Effects of Hyperglycemia and Hypoxia

Abstract

Purpose: To characterize the effect of glutamate receptor activation/inhibition on the secretion of vascular endothelial growth factor (VEGF) in retina-specific glial (Müller) cells under experimental conditions of hyperglycemia and hypoxia, two intrinsic pathologic conditions of diabetic retinopathy.Methods: Purified rat Müller cells were grown in normoglycemic or diabetic-like, hyperglycemic (5.6 or 25 mM glucose, respectively) culture media under normoxic or chemically-induced hypoxic conditions. After treatments, cells were incubated with glutamate receptor agonists and antagonists and VEGF secretion was determined by ELISA. Cell viability was determined by Lactate Dehydrogenase (LDH) secretion-assay and Ki67 immunocytochemistry. Activation of the Akt signal transduction pathway was assessed by western blot using antibodies against phosphorylated Akt. The bio-activity of the secreted VEGF was analyzed by western blot with a phospho-VEGF receptor 2 specific antibody and an in vitro endothelial cell proliferation assay.Results: In control (normoglycemic/normoxic) conditions, N-methyl-D-aspartate receptor (NMDA-R) antagonists MK801 and AP-5 increased secretion of VEGF from Müller cells, and this was not observed after AMPA/kainate receptor blockade. VEGF secretion after NMDA-R antagonists was independent of cell proliferation or cell lysis and it was maintained in cultures grown in hyperglycemia or hypoxia. However, under hyperglycemic and hypoxic conditions, the observed phenomenon was impaired. We also determined that NMDA-R blockade causes a rapid and sustained increase on Akt phosphorylation, a signaling molecule that has been previously linked to VEGF expression. Müller cell-derived VEGF was capable of promoting VEGF receptor 2 phosphorylation and proliferation of endothelial cells.Conclusions: Our results show that NMDA-R exert a tonic inhibition on VEGF secretion in cultures of rat purified Müller cells and indicate that in healthy retina, glutamatergic stimulation could potentially contribute to the protective antiangiogenic role of Müller glia. We suggest that conditions present on diabetic retinopathy could cause malfunction of control points on VEGF synthesis on Müller cells.