Mechanisms of Angiogenesis in Retinopathy of Prematurity

Summary

Principal Investigator: Mary Elizabeth Hartnett
Abstract: Oxygen fluctuations and hyperoxia are risk factors for severe retinopathy of prematurity (ROP). Our hypothesis is that in vivo oxygen stresses, relevant to human severe ROP, cause endothelial apoptosis and disordered angiogenesis through separate pathways to contribute to avascular retina, which precedes and is prerequisite for the development of severe ROP. Specifically, oxygen fluctuations upregulate Mueller cell VEGF and increase VEGF-VEGFR2 signaling in dividing endothelial cells (ECs) at the migrating front to disorient mitotic EC cleavage planes and interfere with developmental retinal angiogenesis, thus contributing to avascular retina. Furthermore, oxygen fluctuations activate EC NADPH oxidase to release reactive oxygen species that trigger apoptosis of ECs and endothelial precursor cells contributing to avascular retina, whereas supplemental oxygen in the setting of oxygen fluctuations further activates EC NADPH oxidase to trigger signaling of cytoskeletal events to disorder cleavage plane orientation in dividing ECs and interfere with normal angiogenesis. We will use oxygen induced retinopathy models (OIR) 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 that mimics the less common form of severe ROP, aggressive posterior ROP (APROP), 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, posterior severe ROP (PSROP). In Specific Aim 1, to determine whether increased VEGF signaling will disorient cleavage planes of dividing ECs at the junction of vascular and avascular retina, we will silence VEGFA or VEGF164 in the rat 50/10 OIR using microRNAs to VEGFA or VEGF164 in an expression vector packaged into a lentiviral vector that will transduce Mueller cells. In Specific Aim 2, we will determine whether differential activation of EC-derived NADPH oxidase triggers different signaling events leading to either apoptosis of ECs or alteration of skeletal events and cleavage plane orientation in dividing ECs. 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 use the 50/10 OIR rescued in supplemental oxygen compared to the standard 50/10 OIR 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 RNA 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 [unreadable]II, 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.
Funding Period: 2003-12-01 - 2010-06-30
more information: NIH RePORT

Top Publications

  1. pmc Reduction in endothelial tip cell filopodia corresponds to reduced intravitreous but not intraretinal vascularization in a model of ROP
    Steven Budd
    Department of Ophthalmology, University of North Carolina Chapel Hill, 103 Mason Farm Road, CB 7041, 6135 NSRB, Chapel Hill, NC 27599, USA
    Exp Eye Res 89:718-27. 2009

Scientific Experts

Detail Information

Publications1

  1. pmc Reduction in endothelial tip cell filopodia corresponds to reduced intravitreous but not intraretinal vascularization in a model of ROP
    Steven Budd
    Department of Ophthalmology, University of North Carolina Chapel Hill, 103 Mason Farm Road, CB 7041, 6135 NSRB, Chapel Hill, NC 27599, USA
    Exp Eye Res 89:718-27. 2009
    ..Reducing the number and length of filopodia/endothelial tip cell may reduce guidance cues for endothelial cells to migrate into the vitreous without interfering with migration into the retina toward a VEGF gradient...