BH4-mediated enhancement of endothelial nitric oxide synthase activity reduces hyperoxia-induced endothelial damage and preserves vascular integrity in the neonate

摘要

Purpose: Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) has important vaso-protective functions that are compromised in the vaso-degenerative phase of retinopathy of prematurity, owing to hyperoxia-induced depletion of the essential NOS cofactor BH4. Since modulating eNOS function can be beneficial or detrimental, our aim was to investigate the effect of BH4 supplementation on eNOS function and vascular regression in hyperoxia.Methods: Endothelial specific eNOS-GFP over-expressing mice at postnatal day-7 (P7) were exposed to hyperoxia for 48 hours in the presence or absence of supplemental BH4, achieved by the administration of sepiapterin, a stable BH4 precursor. Tissue was collected either for retinal flat-mounts that were stained with lectin to determine the extent of vessel coverage or for analysis of BH4 by HPLC, nitrotyrosine (NT) by western blotting, VEGF by ELISA, and NOS activity by arginine to citrulline conversion. Primary retinal microvascular endothelial cells (RMEC) were similarly treated, and hyperoxia-induced damage was determined.Results: Sepiapterin effectively enhanced BH4 levels in hyperoxia-exposed retinas and brains, elevated NOS activity, and reduced NT-modified protein, leading to reversal of the exacerbated vasoregression observed in the presence of eNOS overexpression. In RMEC hyperoxia-mediated depletion of BH4 dysregulated the redox balance by reducing nitrite and elevating superoxide and impaired proliferative ability. BH4 supplementation restored normal RMEC proliferation in vitro and also in vivo, providing a mechanistic link with the enhanced vascular coverage in eNOS-GFP retinas.Conclusion: These results demonstrate that BH4 supplementation corrects hyperoxia-induced RMEC dysfunction and preserves vascular integrity by enhancing eNOS function.