Abstract
Purpose :
Diabetes induces a diabetic retinal neuropathy which precedes and is a biologically plausible cause of diabetic retinopathy, and which is associated with altered availability of a range of retinal neural trophic factors including erythropoietin (EPO). The effect of excess glucose itself on stressed retinal cells is controversial. These investigations aim to assess the effect of "diabetic" glucose levels on hypoxic cultured 661W cells and to characterise the neuroprotective actions of EPO on these cells in a diabetic type environment.
Methods :
661W cells were cultured in serum and insulin free conditions with B27 supplement and pretreated with erythropoietin (EPO) prior to treatment with 25 mM glucose (Gl), the PI3K inhibitor wortmannin or Stat 5 inhibitor as required. 2 hours of hypoxia was induced by 200μM CoCl2 or 10mM EC-oxyrase, and cell health assessed by the cellevent caspase 3/7 activation reagent, annexin V labelling and propridium iodide (PI) staining using immunohistochemistry and flow cytometry. Results are expressed as mean±SE
Results :
Relative to 5 mM Gl, exposure to 25 mM Gl signficantly increased retinal cell death in both models of hypoxia. This effect was predominately apoptotic as indicated by increased caspase 3/7 activation (18%±7% vs 36%±8% cells (P<0.01) and Annexin V staining (20%±4% vs 52%±4 % cells (P<0.01)) in hypoxic cultures in 25mM Gl. EPO reduced this apoptosis in a biphasic manner across a range of concentrations, with 0.04μU/mL EPO reducing the proportion of apoptotic cells to 10%±2% (P<0.01), whilst 2μU/mL EPO did not significantly reduce the apoptosis associated with excess Gl. Conversely, necrosis displayed an opposite biphasic pattern, and was increased from 10%±3% of total hypoxic cells exposed to 25mM Gl with no EPO to 20%±3% with 0.04μU/mL EPO (P<0.05). 2μU/mL EPO had no significant effect on necrosis. Low levels of EPO such as 0.04μU/mL had an overall neuroprotective effect which was inhibited by both 4nM wortmannin and 5μM STAT 5 inhibitor.
Conclusions :
The results indicate that diabetic glucose levels decrease the resilience of 661W photoreceptor-like cells to hypoxia. EPO has a biphasic neuroprotective effect against this glucose induced exacerbation of hypoxic damage in these cells which is lost at higher concentrations and dependent on Stat 5 and PIK3 pathways
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.