This proposal seeks to further study the role of adenosine (ADO) in retinal vasculogenesis using his canine model of retinopathy of prematurity (ROP). While there is a causal association between ROP and exposure to increased amounts of oxygen, the mechanisms are not understood. These studies use the canine model of oxygen-induced retinopathy, which the applicants have demonstrated closely resembles human ROP. The neonatal canine retina is an excellent tissue to study normal developmental vasculogenesis because it is only 60 percent vascularized at birth and, like the human, the primary retinal vasculature develops without mitosis. Exposure of the neonatal dog to hyperoxia for 4 days results in retinal vaso-obliteration, while in both the dog and man, the choroidal vasculature is spared. Upon return to normoxia, neovascularization of the retina occurs similarly to the human ROP. Morphological changes will be examined using ADPase flat-embedded retinas, and functional changes will be assessed and cell types identified using specific histochemical markers. ADO and vascular endothelial growth factor (VEGF), both induced by hypoxia, are the foci of this study. Metabolism of ADO will be elucidated by 5'-nucleotidase histochemistry, ADO immunohistochemistry, and in situ hybridization for expression of 5'-nucleotidase, A1 and A2 ADO receptors. Reaction products will be quantitated morphometrically using image analysis. Expression of VEGF and VEGF receptors will be investigated with in situ hybridization. An inhibitor of 5'-nucleotidase will be used to determine the role of 5'-nucleotidase in both normal and abnormal retinal vasculogenesis. ADO and a potent ADO agonist (NECA) will both be used to determine the role of ADO receptor-mediated events in normal vasculogenesis. In vitro correlates for vasculogenesis and vaso-obliteration will be studied in the dog retinal microvascular endothelial cell preparation. The effects of hypoxia and hyperoxia on proliferation, migration, and tube formation will be studied. The major objective of the studies is to describe and achieve a mechanistic understanding of normal developmental and pathological retinal vasculogenesis and determine whether ADO and/or VEGF are regulators of these processes.
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