Abstract
Purpose :
Increased intracellular generation of reactive oxygen species (ROS) plays a major role in the pathology of diabetic retinopathy, and phagocyte-like NADPH oxidase-2 (Nox2) is considered as one of the major sources of cytosolic ROS.
Activation of Rac1, a member of Nox2 holoenzyme, is regulated by post-translational prenylation at its C-terminal cysteine. Rac1 activation is also regulated via precise interplay between its regulatory factors including guanine nucleotide-dissociation inhibitor (GDI) and guanine nucleotide exchange factors (Vav2). The aim of this study was to investigate the putative mechanisms underlying the activation of Rac1 in the development of diabetic retinopathy.
Methods :
The levels of protein farnesyl-transferase (FNTA), GDI and Vav2 were quantified in the human retinal endothelial cells (HRECs), incubated in normal (NG) or high (HG) glucose for 96 hours. Role of prenylation in the regulation of Rac1-Nox2-ROS signaling was confirmed in the HRECs transfected with FNTA-siRNA. Rac1 activity was measured by G-LISA, Nox2 activity by a lucigenin-based method, and ROS by DCFDA method. Key parameters of Rac1 activation were quantified in the retinal microvasculature from human donors with documented diabetic retinopathy.
Results :
High glucose increased the expression of FNTA and Vav2, and decreased GDI. Inhibition of prenylation of Rac1 by FTNA-siRNA ameliorated glucose-induced increase in Rac1 and Nox2 activities, and decreased ROS levels. Furthermore, compared to the age-matched nondiabetic human donors, retinal microvessels from donors with diabetic retinopathy had increased FNTA levels, and an activated Rac1-Nox2-ROS pathway.
Conclusions :
Post-translational prenylation of Rac1 contributes to its activation and Nox2-mediated generation of cytosolic ROS in diabetes. Regulation of Rac1 activation in early stages by targeting its prenylation should help impede ROS accumulation, and inhibit the development of diabetic retinopathy.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.