Rat retinal endothelial cell dysfunction induced by hyperglycemia and oxidative stress: additive effects?

Abstract

Abstract: : Purpose:High blood glucose levels play a major role in Diabetic Retinopathy. Moreover, free radical damage appears to be involved in this pathology. This work aimed to investigate if hyperglycemia and oxidative stress cause retinal endothelial cell dysfunction and if cells exposed to high glucose levels are less resistant to stress conditions. Methods: Rat retinal capillary endothelial cell line (TR–iBRB2) was cultured in DMEM with 10% FCS. Cells were exposed to high concentrations of glucose or mannitol for 7 days. In addition, control or high glucose–treated cells were exposed to NOC–18 (NO donor) or H2O2 for 24h. Cell viability was assessed by MTT assay and cell proliferation was determined by [methyl–³H] thymidine uptake. Apoptotic cells were identified by Hoechst assay and pro–caspase 3 levels were quantified by western blot. Results: Exposure of retinal endothelial cells with high glucose concentrations (7 days) significantly decreased cell viability. Exposure of cells to mannitol, used as an osmotic control, did not cause a toxic effect. However, high glucose and mannitol decreased endothelial cell proliferation. In addition, pro–caspase 3 expression was not significantly altered in cells exposed to high glucose levels, although the number of apoptotic cells increased. Both NOC–18 and H₂O₂ decreased cell viability and that effect was additive when cells were incubated with high glucose levels. Apoptotic cells also increased when cells were exposed to NOC–18 or H₂O₂, but hyperglycaemia, in those conditions, did not alter the number of apoptotic cells. Conclusions: These results show that hyperglycemia has a dual effect: increases cell degeneration and decreases cell proliferation, but it is not clear whether hyperglycemia increases the susceptibility of cells to stress conditions.

Keywords: diabetic retinopathy • oxidation/oxidative or free radical damage • cell death/apoptosis