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Denise M McDonald, Kevin S Edgar, Tom A Gardiner, Zvonimir S Katusic; BH4 supplementation improves vascular integrity during hyperoxia in oxygen induced retinopathy. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5376.
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The initiating insult in retinopathy of prematurity (ROP) is hyperoxia-induced oxidative-nitrosative stress and vascular damage. Dysfunctional endothelial nitric oxide synthase (eNOS) plays an important role in contributing to this vascular stress. Normally, eNOS-derived NO is vasoprotective, a function which is dependent upon the availability of the cofactor, tetrahydrobiopterin (BH4). In disease, however, eNOS can become dysfunctional, producing the oxygen free radical superoxide in preference to NO causing a shift in the nitroso-redox balance towards one which is detrimental to vascular health. Previously, we have shown that, in hyperoxia, there is insufficient BH4 levels to meet the demands of enhanced eNOS activity in an endothelial specific eNOSGFP transgenic model, which resulted in exacerbated vascular closure. Here our aim was to determine if supplementing BH4 levels can reverse this trend and protect the retina from hyperoxic insult.
BH4 supplementation was achieved by the addition of sepiapterin, a synthetic pterin that is converted intracellularly to BH4 via the pterin salvage pathway. ENOSGFP mice and wild type (WT) litter mate controls were treated with sepiapterin or vehicle control (C) at postnatal day 7 (P7) and subjected to 2 days of hyperoxia. Upon removal of the eyes retinal flat-mounts were stained with lectin and viewed by confocal microscopy to determine the extent of vessel closure. The second eye was prepared for analysis of BH4 by hplc, VEGF by ELISA, and nitrotyrosine (NT) by western blotting as a surrogate for peroxynitrite levels; these indicators were correlated with the degree of vascular closure.
Assay in brain and retinal tissue confirmed the efficiency of sepiapterin treatment to elevate BH4 levels. In the WT group BH4 supplementation had no effect on vascular closure (25% vaso-obliterated area). However, in agreement with our hypothesis, in the eNOSGFP group, BH4 supplementation returned vaso-obliterated areas (35%) to levels found in the WT group (25%) which correlated with reduced retinal NT immunoreactivity, indicative of improved NO availability and decreased oxidative free radical production. VEGF levels were unaltered throughout.
Together our results show that supplementing BH4 levels, improves NO bioavailability, decreases oxidative free radical production and positively impacts on vascular integrity in hyperoxia.
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