Abstract
Purpose::
Nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) in retinal glial cells, has anti-angiogenic effects on restorative retinal vascularization following ischemia and exacerbates intra-vitreal neovascularization (NV). Future therapeutic strategies which result in the inhibition of iNOS in the ischemic retina may permit intra-retinal vascular recovery. The activity of iNOS is dependent on the presence of GTPCH, the rate limiting enzyme responsible for the production of the NOS co-factor BH4. In the immune system iNOS and GTPCH are transcriptionally co-induced by proinflammatory cytokines such as TNFα. The aim of this study was to determine if TNFα is required to induce iNOS protein expression and if it is also involved in regulating GTPCH levels in an animal model of oxygen induced retinopathy (OIR).
Methods::
iNOS -/- and TNFα -/- animals were subjected to OIR by exposure of neonatal mice to 75% oxygen between postnatal days 7 and 12 (P7-P12). The animals were returned to room air at P12 and the retinas removed at P12, P13 and P17 time points. Eyes were snap frozen and the retinas removed for RNA extraction. Following reverse transcription quantitative RT-PCR was performed using a Lightcycler, SYBR green I and mouse specific primers. Data was normalized to the expression of 18S and 28S ribosomal RNA. Expression of iNOS and peroxynitrite was also analysed by Western blotting.
Results::
iNOS mRNA expression was significantly increased (12 fold) in TNFα-/- animals at P13 and P17; in contrast GTPCH expression was moderately increased (2 fold) at each of these time points. iNOS immunoreactivity was negligible at P12 but increased significantly at P13 in all groups. These changes coincided with a decrease in the production of peroxynitrite in the TNFα-/- animals compared to controls.
Conclusions::
We have shown that in TNFα depleted animals there is a significant stimulation of iNOS expression and a moderate increase in GTPCH expression. We have also shown that in the absence of TNFα there is a reduction in hypoxia induced peroxynitrite despite the presence of significant amounts of iNOS. Our results suggest that while the expression of iNOS is driven by tissue hypoxia, TNFα may play a role in stimulating GTPCH expression and thus full iNOS activity in the retina.
Keywords: retinal neovascularization • ischemia • nitric oxide