Abstract
Purpose:
We have shown that deletion of arginase 2 (A2) limits vascular injury in the mouse model of oxygen-induced retinopathy (OIR) by reducing hyperoxia-induced vaso-obliteration. This protective effect is associated with decreased peroxynitrite formation and reduced activation of microglia/macrophage cells (ARVO, 2012). We have now examined the involvement of NOS uncoupling and A1 expression in this process.
Methods:
Neonatal mice [lacking one copy of A1 (A1+/-), lacking both copies of A2 (A2-/-), and wild type (WT)] were maintained in hyperoxia (70% oxygen) from postnatal day 7 (p7) to p12 and then returned to normoxia. Controls were maintained in normoxia. Mice were sacrificed at different times. Retinas were processed for analysis of NOS uncoupling by using diaminofluorescein (DAF) imaging of nitric oxide (NO) and dihydroethidium (DHE) imaging of superoxide (O2.-). Retinal samples were also processed for immunolocalization and western blotting analysis of expression of arginase 1 and activation of survival pathways in relation to retinal vascular injury and repair.
Results:
Formation of NO was reduced by 60% in the hyperoxia-treated WT retina as compared with the normoxia control (P<0.05). NO formation was preserved in the A2-/- OIR retina (P<0.05). Formation of O2.- was increased by 100% in WT OIR retinas as compared with WT controls after 24 hours of hyperoxia (P<0.05). This effect was blocked by treatment with the NOS inhibitor (L-NAME) or by A2 deletion (P<0.05). The vaso-protective effects of A2 deletion in the OIR retina were associated with marked up-regulation of A1immunoreactivity. Furthermore, hyperoxia-induced vaso-obliteration was markedly enhanced in the A1+/- OIR retina as compared with WT OIR retina (P<0.05). These protective effects of A2 deletion and A1 upregulation were accompanied by increased formation of the autophagic marker microtubule associated protein 1 light chain 3 and activation of ERK and Akt survival pathways (P<0.05).
Conclusions:
Arginase 2 deletion prevents hyperoxia-induced retinal vascular injury by attenuating NOS uncoupling mediated superoxide formation which is associated with upregulation of A1 expression and activation of autophagy and survival pathways.
Keywords: 634 oxidation/oxidative or free radical damage •
617 nitric oxide •
449 cell survival