Purpose:Hyperoxia is a dominant pathogenic factor in retinopathy of prematurity (ROP) resulting in an arrest of normal retinal vascular development accompanied by vaso-obliteration of pre-established vessels. The increased generation of endothelial cell-derived reactive oxygen species (ROS) and reactive nitrogen species (RNS) are associated with vessel loss during hyperoxia; in particular, dysregulated endothelial nitric oxide synthase (eNOS) is implicated in aberrant generation of such effectors in animal models of ROP. Defining the key pathways involved in vaso-obliteration will aid the discovery of therapeutic targets that protect the retina from hyperoxia-induced vascular damage. Therefore we established a model system to investigate the effect of hyperoxia and eNOS over-expression on retinal microvascular endothelial cells in vitro, examining nitrosative stress, cell death via apoptosis and cellular senescence. Methods:Primary retinal microvascular endothelial cells (RMECs) were exposed to physiologically relevant 6% oxygen, standard cell culture incubator conditions (21% oxygen) and 70% oxygen. Apoptosis was quantified by nuclear morphology, active caspase-3 and TUNEL staining. Mechanisms of cell injury were investigated using inhibitors and scavengers of superoxide and of peroxynitrite and nitrotyrosine quantified by western blotting. Senescence was determined by β-galactosidase staining the co-localisation of γH2AX and 53BP-1 nuclear foci, and the formation of multiple nuclei. eNOS over-expression was achieved by viral infection with an adenovirus encoding eNOS-GFP and compared to control, GFP expressing virus. Results:24 hour hyperoxia treatment of RMECs increased nitrotyrosine formation and apoptosis. Inhibition of either peroxynitrite or inhibition of superoxide reduced hyperoxia-mediated apoptosis. In addition, hyperoxia induced a significant increase in the senescence markers β-galactosidase and 53BP-1. eNOS over-expression further increased apoptosis under hyperoxic conditions. Conclusions:Collectively, our results demonstrate that hyperoxia increases cellular senescence and cell death via apoptosis in RMECs. This apoptosis can be reduced through inhibition of ROS and RNS. eNOS over-expression increased RNS production, leading to a further increase in hyperoxia induced apoptosis.