Renu A Kowluru1, Saiyeda Noor Abbas. 1. Kresge Eye Institute, Wayne State University, 4717 St. Antoine, Detroit, MI 48201, USA. rkowluru@med.wayne.edu
Abstract
PURPOSE: Oxidative stress is increased in the retina in diabetes, and antioxidants inhibit activation of caspase-3 and the development of retinopathy. The purpose of this study was to investigate the effect of diabetes on the release of cytochrome c from mitochondria and translocation of Bax into mitochondria in the rat retina and in the isolated retinal capillary cells. METHODS: Mitochondria and cytosol fractions were prepared from retina of rats with streptozotocin-induced diabetes and from the isolated retinal endothelial cells and pericytes incubated in 5 or 20 mM glucose medium for up to 10 days in the presence of superoxide dismutase (SOD) or a synthetic mimetic of SOD (MnTBAP). The release of cytochrome c into the cytosol and translocation of the proapoptotic protein Bax into the mitochondria were determined by the Western blot technique and cell death by caspase-3 activity and ELISA assay. RESULTS: Diabetes of 8 months' duration in rats increased the release of cytochrome c into the cytosol and Bax into the mitochondria prepared from the retina, and this phenomenon was not observed at 2 months of diabetes. Incubation of isolated retinal capillary cells with 20 mM glucose increased cytochrome c content in the cytosol and Bax in the mitochondria, and these abnormalities were accompanied by increased cell apoptosis. Inclusion of SOD or its mimetic inhibited glucose-induced release of cytochrome c, translocation of Bax, and apoptosis. CONCLUSIONS: Retinal mitochondria become leaky when the duration of diabetes is such that capillary cell apoptosis can be observed; cytochrome c starts to accumulate in the cytosol and Bax into the mitochondria. Inhibition of superoxides inhibits glucose-induced release of cytochrome c and Bax and inhibits apoptosis in both endothelial cells and pericytes. Identifying the mechanism by which retinal capillary cells undergo apoptosis may reveal novel therapies to inhibit the development of retinopathy in diabetes.
PURPOSE: Oxidative stress is increased in the retina in diabetes, and antioxidants inhibit activation of caspase-3 and the development of retinopathy. The purpose of this study was to investigate the effect of diabetes on the release of cytochrome c from mitochondria and translocation of Bax into mitochondria in the rat retina and in the isolated retinal capillary cells. METHODS: Mitochondria and cytosol fractions were prepared from retina of rats with streptozotocin-induced diabetes and from the isolated retinal endothelial cells and pericytes incubated in 5 or 20 mM glucose medium for up to 10 days in the presence of superoxide dismutase (SOD) or a synthetic mimetic of SOD (MnTBAP). The release of cytochrome c into the cytosol and translocation of the proapoptotic protein Bax into the mitochondria were determined by the Western blot technique and cell death by caspase-3 activity and ELISA assay. RESULTS:Diabetes of 8 months' duration in rats increased the release of cytochrome c into the cytosol and Bax into the mitochondria prepared from the retina, and this phenomenon was not observed at 2 months of diabetes. Incubation of isolated retinal capillary cells with 20 mM glucose increased cytochrome c content in the cytosol and Bax in the mitochondria, and these abnormalities were accompanied by increased cell apoptosis. Inclusion of SOD or its mimetic inhibited glucose-induced release of cytochrome c, translocation of Bax, and apoptosis. CONCLUSIONS: Retinal mitochondria become leaky when the duration of diabetes is such that capillary cell apoptosis can be observed; cytochrome c starts to accumulate in the cytosol and Bax into the mitochondria. Inhibition of superoxides inhibits glucose-induced release of cytochrome c and Bax and inhibits apoptosis in both endothelial cells and pericytes. Identifying the mechanism by which retinal capillary cells undergo apoptosis may reveal novel therapies to inhibit the development of retinopathy in diabetes.