Abstract
AbstractPurposeTo elucidate the role of endothelial nitric oxide synthase (eNOS) activation in the induction of vascular endothelial growth factor (VEGF)‐induced permeability, the underlying mechanism and the contribution to exaggerated vascular leakage in eye disease.MethodsRetinal neovascularization and pathological edema in the superficial and deep retinal vascular plexa were investigated using the oxygen‐induced retinopathy (OIR) mouse model. eNOS activity was blocked either chemically by using the NO inhibitor or genetically by using mutant mice (Mut) in which serine 1176 in eNOS is replaced by alanine (Nos3S1176A/S1176A,therefore is unable to induce eNOS activity). Retina avascular area and proliferative vessel tuft formation were assessed at postnatal (P) day 17. Leakage was determined by injecting 25 nm fluorescent microspheres in the circulation followed by microscopy. Isolectin B4, eNOS, vascular endothelial cadherin (VEC), and phosphoY685‐VEC immunostaining was assessed by confocal imaging followed by image analysis using ImageJ. Students t‐test was used for statistical evaluation. p < 0.05 was regarded as statistically significant.ResultsAdministration of the NO inhibitor during P12‐P17 reduced overall pathological tuft area (p < 0.05), and decreased the area of individual tufts (p < 0.01) compared to control. Mut displayed reduced neovascular tuft formation (p < 0.01) and decreased tuft size compared to wildtype mice (WT) (p < 0.05). Mice unable to activate eNOS displayed a junctional phenotype. Firstly, phosphoY685‐VEC in tufts was decreased in Mut compared to WT (p < 0.05). Secondly, immunostaining for eNOS colocalized with VEC in vessels of WT but not in Mut (p < 0.05). Thirdly, vascular leakage was reduced in OIR‐challanged Mut (p < 0.01).ConclusionOur data establish that eNOS is critical for VEC‐regulated endothelial junction stability and vascular leakage. Thus, eNOS is an important target for the development of therapeutic agents to treat eye disease.
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