Oxygen fluctuations and increased inspired oxygen (hyperoxia) are risk factors for severe retinopathy of prematurity (ROP). Our hypothesis is that in vivo oxygen stresses, relevant to human severe ROP, increase avascular retina, which precedes and is a necessary prerequisite for the development of severe ROP. The increased avascular retina occurs through two different events: disordered angiogenesis and endothelial apoptosis. Specifically, oxygen fluctuations and relative tissue hypoxia upregulate Mueller cell VEGF and increases VEGF-VEGFR2 signaling in dividing endothelial cells (ECs) at the migrating front to disorient mitotic EC cleavage planes and interfere with normal retinal angiogenesis, thus increasing peripheral avascular retina. Furthermore oxygen fluctuations or hyperoxia cause different degrees of activation of NADPH oxidase to release reactive oxygen species that trigger apoptosis of ECs and endothelial precursor cells reducing retinal vascular development and increasing avascular retina, whereas supplemental oxygen in the setting of oxygen fluctuations will further activate NADPH oxidase to trigger signaling of cytoskeletal events to disorder cleavage plane orientation and interfere with normal angiogenesis. We will use oxygen induced retinopathy (OIR) models in rodents, which undergo retinal vascular development after birth. We will use either the genetically-manipulable mouse OIR model to study mechanisms of hyperoxia or relative tissue hypoxia, or the rat 50/10 OIR model in which the oxygen fluctuations mimic those experienced by preterm human infants that develop the more common form of severe ROP (zone II, stage 3 ROP). In Specific Aim 1, to test the prediction that increased VEGF signaling will disorient cleavage planes of dividing endothelial cells at the junction of vascular and avascular retina and interfere with intraretinal vascularization, we will silence Mueller cell derived VEGF164 in the rat 50/10 OIR using a microRNA to VEGFA or VEGF164 in an expression cassette packaged into a lentiviral vector. In Specific Aim 2, we will test the prediction that differential activation of NADPH oxidase triggers different signaling events leading to either apoptosis of endothelial cells or alteration of skeletal events and cleavage plane orientation in dividing endothelial cells. To do this we will use knockout mice to p47phox, a subunit of NADPH oxidase or pharmacologic inhibitors of NADPH oxidase. We will study the effects of EC-derived NADPH oxidase by depleting animals of macrophages. To change the degree of activation of NADPH oxidase, we will also use the 50/10 OIR model rescued in hyperoxia compared to the standard 50/10 OIR model rescued in room air. Methods include: confocal microscopy of retinal flat mounts to visualize endothelial cells at the junction of vascular and avascular retina to quantify apoptotic cells, capillary density, vascular and avascular retina, and to count the number of random mitotic planes of dividing phospho-histone stained endothelial cells;cryosections for phosphorylated VEGFR1 and 2, apoptosis (TUNEL, cleaved caspase-3) of co-labeled cells;laser capture microdissection;real-time-PCR to quantitate and in situ hybridization to detect location of mRNA of VEGF isoforms, VEGF receptors 1 and 2, neuropilins;ELISA and Western blot to measure protein (VEGF;cleaved caspase-3);immunoprecipitation and probing to detect phosphorylated VEGF receptors, protein kinase 2II, JAKs, STATs;construction of microRNAs to VEGFA or VEGF164 packaged into lentiviral vectors with a CD44 promoter;subretinal injections;systemic NADPH oxidase inhibitors or clodronate;measurement of reactive oxygen species (e.g., dihydroethidium);and NADPH oxidase activation. PUBLIC HEALTH RELEVANCE: The Institute of Medicine reported that preterm birth is a growing public health problem that costs society at least $26 billion each year (http://www.iom.edu/Object.File/Master/35/975/pretermbirth.pdf). Between 1981 and 2006, there was a 30% increase in preterm births, now accounting for 12.5% of all births in the U.S. Furthermore, with greater ability to save infants of younger gestational age and smaller birth weights, there is greater risk of vision loss and total blindness from severe retinopathy of prematurity (ROP). Limiting VEGF, reducing inspired oxygen, and quenching reactive oxygen species (all suggested in the past as therapies for ROP) each can have detrimental effects on the developing infant. Our proposal seeks to understand the mechanisms of severe ROP in order to develop future safer, less destructive, and more effective therapies than currently available.