Abstract
Purpose:
Retinal mitochondria become dysfunctional at duration of diabetes when capillary cell apoptosis (a phenomenon that precedes the development of pathology characteristic of diabetic retinopathy) is observed. However, an overall increase in oxidative stress in the retina and its capillary cells can be seen before mitochondrial dysfunction. In addition to mitochondria, NADPH oxidase (Nox) has an important role in generation of intracellular ROS, and we have shown that Rac1-mediated Nox-2 is activated in the retina in diabetes. The aim of this research is to investigate the relationship between the activation of Nox2 and mitochondrial damage.
Methods:
Total ROS levels, Rac1 activation (GTP-bound), and Nox-2 activity (by luminescence assay) were determined in the retinal endothelial cells incubated in 5mM or 20mM glucose for 3 hours to 96 hours. Mitochondrial damage was evaluated by quantifying its DNA damage (extended length PCR), and capillary cell apoptosis was determined by an ELISA method. Similar measurements were made in the retina from rats/mice diabetic for 15 days to ~8 months.
Results:
In retinal endothelial cells, glucose insult for 3-24 hours increased ROS levels and activated Rac1 and Nox-2, but mitochondrial damage and cell apoptosis were not observed till the duration of high glucose exposure was extended beyond 48 hours. Consistent with these in vitro results, increase in overall ROS levels and activation of Rac1 and Nox-2 in the retina were observed within 15 days of diabetes, but mitochondrial damage and capillary cells apoptosis (TUNEL-positive cells in retinal vasculature) were seen only after 6 months of diabetes.
Conclusions:
In the pathogenesis of diabetic retinopathy, activation of Nox-2 is an early signaling event in increasing cytosolic ROS. These ROS, in turn, initiate the damage of mitochondria and its electron transport (ETC) system. Thus, prevention of Nox-2 activation has potential to prevent mitochondrial damage and the initiation of the vicious cycle of superoxide production initiated by dysfunctional ETC system.
Keywords: 499 diabetic retinopathy •
600 mitochondria •
634 oxidation/oxidative or free radical damage