June 2013
Volume 54, Issue 15
ARVO Annual Meeting Abstract  |   June 2013
Mechanisms of Hyperoxia-induced Retinal Endothelial Cell Death and Senescence
Author Affiliations & Notes
  • Orla Galvin
    Queen's University Belfast, Belfast, United Kingdom
  • Tom Gardiner
    Queen's University Belfast, Belfast, United Kingdom
  • Denise McDonald
    Queen's University Belfast, Belfast, United Kingdom
  • Footnotes
    Commercial Relationships Orla Galvin, None; Tom Gardiner, None; Denise McDonald, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 5573. doi:
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      Orla Galvin, Tom Gardiner, Denise McDonald, McDonald; Mechanisms of Hyperoxia-induced Retinal Endothelial Cell Death and Senescence. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5573.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: 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.

Methods: 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.

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.

Keywords: 426 apoptosis/cell death • 635 oxygen • 706 retinopathy of prematurity  

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