Hyperoxia is a dominant pathogenic factor in retinopathy of prematurity (ROP) resulting in arrest of normal vascular development and vaso-obliteration of pre-established retinal 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 investigated 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.
Primary retinal microvascular endothelial cells (RMECs) were exposed to 6%O2, standard cell culture incubator conditions (21%O2) and 70%O2. 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, γH2AX and 53BP-1 staining. eNOS over-expression was achieved by viral infection with an adenovirus encoding eNOS-GFP and compared to control, GFP expressing virus.
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.
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
|Journal||Investigative Opthalmology and Visual Science|
|Publication status||Published - Jun 2013|