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A. Santiago, A.J. Cristóvão, P.F. Santos, A.F. Ambrósio; High Glucose–Induced Apoptosis in Cultured Retinal Neural Cells Is Dependent on Apoptosis–Inducing Factor . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4239.
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© ARVO (1962-2015); The Authors (2016-present)
Diabetic retinopathy (DR) is a leading cause of blindness in the western countries. Several reports show that retinal neural cells dye by apoptosis during diabetes. Previously, we observed an increase in TUNEL–positive cells in high glucose–treated cells without an increase in caspase–3–like enzymes activity, suggesting that in these conditions apoptosis is independent of caspase–3. Apoptosis–inducing factor (AIF) is a flavoprotein usually located in the mitochondrial intermembrane space, which upon lethal signalling translocates to the nucleus, where it binds to DNA and causes chromatin condensation. In this work, we investigated whether AIF is involved in the increase in apoptosis in cultured retinal neural cells exposed to elevated glucose concentration.
Rat retinal neural cells were cultured with 30 mM glucose or 25 mM mannitol (osmotic control) for 7 days. Cells undergoing apoptosis, with phosphatidylserine in the outer layer of plasma membrane, were visualized with Annexin–V staining. To identify cells with active caspases, cells were incubated with a fluorescent inhibitor of active caspases, FAM–VAD–FMK. Changes in mitochondrial and nuclear levels of AIF were studied by Western Blotting.
Elevated glucose induced a significant increase in the number of Annexin–V positive cells, suggesting an increase in apoptosis. However, the number of cells with active caspases did not increase significantly in high glucose–treated cells. Mitochondria AIF levels decreased in high glucose–treated cells, comparing to control. Concomitantly, AIF levels increased in the nuclear fraction of high glucose–treated cells. Mannitol did not cause significant changes as compared to control, indicating that high glucose–induced apoptosis is not due to changes in osmolarity.
These results show that elevated glucose increases retinal neural cell apoptosis, supporting previous findings of neurodegeneration during DR. Also, in this in vitro model, high glucose–induced apoptosis is mediated through AIF, independently of caspases activation.
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