The Role of the Nicotinic Cholinergic Pathway in Retinopathy of Prematurity

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DESCRIPTION (provided by applicant): Retinopathy of prematurity (ROP), is a leading cause of vision impairment and blindness in children in the United States, due to pathological retinal neovascularization. We have discovered a novel angiogenic pathway that is involved in pathological neovascularization. This pathway is mediated by an endothelial nicotinic acetylcholine receptor (nAChR). The endothelial nAChR is a ligand-gated cationic channel that is activated by the endogenous signaling molecule, acetylcholine (ACh). Activation of this receptor induces endothelial cell mitogenesis, migration and tube formation and promotes angiogenesis. The pathway is upregulated by hypoxia, and by other angiogenic factors such as the vascular endothelial growth factor (VEGF). Accordingly, we propose the following Specific Aims to develop a novel therapy for ROP: 1. Characterize the endothelial nicotinic cholinergic pathway in the developing retina, and determine its role in normal vascularization. We hypothesize that normal development of the retina will not be adversely affected by pharmacological antagonism of the EC nAChRs. This hypothesis is based in part on the normal phenotype of the a7-nAChR deficient mouse. We will begin by determining, during normal development, the level of expression and localization of key elements of the nAChR pathway including the high affinity choline transporter, choline acetyltransferase, and the nAChR subunits. We will study EC from normal retinal vessels using laser capture microdissection and real time RT-PCR, in situ hybridization and immunohistochemistry. We will carefully assess vascular and neuronal development in the retina of the a7-nAChR deficient mouse. Finally, we will determine if pharmacological antagonism of nAChRs in the eye (comparing non-selective versus a7-nAChR selective antagonists) will adversely effect normal neuronal or vascular development. 2. Determine if excessive activation of this pathway contributes to retinal neovascularization in a murine model of ROP, using pharmacological and genetic knockdown of EC nAChRs. Using the methods described above, we will determine if retinal neovascularization is associated with increased retinal expression of any components of the nAChR pathway. We will determine if administration of the non-selective (mecamylamine) or a7-preferential (methyllycaconitine) nAChR antagonists suppress retinal neovascularization. The effect of nAChR antagonists on retinal neuronal function will be assessed by ERGs. ROP will be induced in a7- nAChR knockout mice and littermate controls to determine if signaling through a7- nAChRs contributes to retinal neovascularization. We will perform biochemical studies to assess the relationship between plasma and retinal levels of ACh, and correlate these levels to changes in retinal levels of vascular endothelial growth factor (VEGF), retinal vascularity and permeability. PUBLIC HEALTH RELEVANCE: Abnormal blood vessel formation is a risk factor in retinopathy of prematurity. Retinopathy of prematurity (ROP) is a significant cause of vision impairment and blindness in more than 50% of premature infants as a result of abnormal blood vessel formation in the retina. The possible risk factors for ROP are the oxygenation of these infants after birth and the accumulation of growth factors that are responsible for the formation of abnormal blood vessels. In this study, we intend to understand the role of a particular receptor in these abnormal vessels and develop a new therapy to prevent visual loss and blindness in preterm infants.