Blockade of NOX2 or arginase prevents diabetes-induced retinal endothelial cell senescence by restoring NO bioavailability

Abstract

Purpose : In diabetes, decreased bioavailability of the signaling molecule and endothelial cell (EC) specific vasodilator nitric oxide (NO) plays a major role in EC dysfunction. Diabetes can reduce NO by increasing superoxide (O₂^.-) which reacts with NO to form peroxynitrite and by increasing the activity of the ureohydrolase enzyme arginase (Arg). Arg competes with NO synthase (NOS) for its substrate L-arginine causing NOS to produce more O₂^.- and less NO. ROS and decreased NO can cause premature senescence of ECs leading to defective vascular repair. Our previous work has demonstrated the role of the O₂^.- generating enzyme NOX2, decreased NO and overactive Arg in diabetic retinopathy. Here we investigated the role of ROS, Arg and decreased NO in diabetes-induced EC senescence

Methods : Studies were performed using diabetic wild type and NOX2^-/- mice and retinal ECs treated with 25 mM glucose with or without NOX2 blocking peptide gp91phox. Other ECs were treated with H₂O₂ or the NOS inhibitor L-NAME with or without the NO donor SNAP or the arginase inhibitor ABH. Expression of NOX2 and Arg was assessed by western blot and immunofluorescence. Arginase activity was assayed by measuring urea formation. ROS formation was measured by dihydroethidium and dichlorofluorescein assays. NO formation was measured using chemiluminescence to assay nitrite formation. Cellular senescence was determined by assaying senescence associated β-galactosidase (SA- β-gal) activity

Results : Retinal cells from diabetic mice and high glucose-treated retinal ECs showed significant increases in SA-β-gal activity along with increased ROS formation, elevated Arg activity and expression and decreased NO formation. Deletion of NOX2 or specific blockade of its activity prevented increases in ROS production, normalized arginase activity and expression, restored NO formation and reduced SA-β-gal activity. L-NAME or H₂O₂ also induced retinal EC senescence which was prevented by treatment with the NO donor or the arginase inhibitor

Conclusions : These results indicate that decreased NO bioavailability accelerates EC senescence. NOX2-generated ROS contribute importantly to diabetes/high glucose induced activation of Arg and decreased NO. Blockade of NOX2 or Arg represents a strategy to prevent diabetes-induced premature EC senescence and promote healthy vascular repair

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.