High Glucose Increases Vascular Endothelial Growth Factor (VEGF) Secretion in Human Müller Cells (MIO-M1) and Human Organotypic Retinal Cultures (HORCs)

Abstract

Purpose : Diabetic retinopathy (DR) is a major cause of vision loss. In DR, elevated blood glucose and hypoxia cause release of VEGF which contributes to macular oedema and neovascularization of the retina. The aim of these experiments was to investigate the effects of raised glucose and hypoxia on VEGF release from the human Müller (MIO-M1) cells and human organotypic retinal cultures (HORCs).

Methods : MIO-M1 cells were routinely cultured in DMEM 10% FBS. Human tissue was provided by the East Anglian Eye Bank within 24 hours post mortem, with full ethical approval and consent. Five retinal explants (HORCs), 4mm in diameter, were taken from the paramacular region of each retina. For experimental conditions, MIO-M1 cells or HORCs were cultured in serum free DMEM in normal (5.5mM) or high glucose (25mM; HG). Oxygen deprivation was induced using a custom-made, computer-controlled, environmental chamber. Cell viability/death was assessed by MTS and/or LDH assays and RGC loss by NeuN immunohistochemistry. VEGF mRNA levels were measured using QRT-PCR and secretion by ELISA. All experiments were carried out to at least n=4.

Results : Exposure of MIO-M1 cells and HORCs to HG for 24hrs under normoxia did not alter LDH release. The viability of MIO-M1 cells was comparable to control, and NeuN-labelled RGCs in HORCs showed a similar number to control with HG for 24hrs. HG treatment caused a 4-fold increase (p<0.05) in expression of VEGF mRNA in MIO-M1 at 24hrs. VEGF secretion in MIO-M1 cells exposed to HG increased with time, with a 2.6-fold increase (p<0.05) at 72hrs. A 4.5-fold increase (p<0.05) was also seen with 20mM glucose in HORCs at 24hrs. Exposure of MIO-M1 to hypoxia (0%O₂) showed at large time-dependent increases in VEGF release under control and HG conditions; increases reached 40-fold at 72hrs (p<0.05). The PKCβ inhibitor LY333531 (1µM) reduced VEGF secretion under HG/hypoxia at 24hrs (p<0.05).

Conclusions : VEGF secretion is increased with raised glucose in both MIO-M1 cells and human retinal explants. Inhibition of VEGF secretion by LY333531 supports a role for PKCβ inhibitors in treatment of DR.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.