Background:Hypoxia and consequent production of vascular endothelial growth factor A (VEGFA)promote blood vessel leakiness and edema in ocular diseases. Anti-VEGFA therapeutics mayaggravate hypoxia; therefore, therapy development is needed.Methods:Oxygen-induced retinopathy was used as a model to test the role of nitric oxide (NO) inpathological neovascularization and vessel permeability. Suppression of NO formation wasachieved chemically using L-NMMA, or genetically, in endothelial NO synthase serine to alanine(S1176A) mutant mice.Results:Suppression of NO formation resulted in reduced retinal neoangiogenesis. Remainingvascular tufts exhibited reduced vascular leakage through stabilized endothelial adherens junctions,manifested as reduced phosphorylation of vascular endothelial (VE)-cadherin Y685 in a c-Src-dependent manner. Treatment with a single dose of L-NMMA in established retinopathy restoredthe vascular barrier and prevented leakage.Conclusions:We conclude that NO destabilizes adheren junctions, resulting in vascularhyperpermeability, by converging with the VEGFA/VEGFR2/c-Src/VE-cadherin pathway.Funding:This study was supported by the Swedish Cancer foundation (19 0119 Pj ), the SwedishResearch Council (2020-01349), the Knut and Alice Wallenberg foundation (KAW 2020.0057) and aFondation Leducq Transatlantic Network of Excellence Grant in Neurovascular Disease (17 CVD03). KAW also supported LCW with a Wallenberg Scholar grant (2015.0275). WCS was supportedby Grants R35 HL139945, P01 HL1070205, AHA MERIT Award. DV was supported by grants fromthe Deutsche Forschungsgemeinschaft, SFB1450, B03, and CRU342, P2. [ABSTRACT FROM AUTHOR]