The Role of the Nicotinic Cholinergic Pathway in Retinopathy of Prematurity

描述（由申请人提供）：早产儿视网膜病变（ROP）是美国儿童视力损害和失明的主要原因，原因是病理性视网膜新生血管。我们发现了一种新的血管生成途径，它与病理性新生血管形成有关。该途径由内皮尼古丁乙酰胆碱受体（nAChR）介导。内皮细胞nAChR是一种配体门控阳离子通道，可被内源性信号分子乙酰胆碱（ACh）激活。该受体的激活诱导内皮细胞有丝分裂、迁移和管的形成，并促进血管生成。缺氧和其他血管生成因子（如血管内皮生长因子（VEGF））可上调该通路。因此，我们提出以下具体目标来开发一种新的治疗ROP的方法：1。描述发育中的视网膜内皮烟碱能通路，并确定其在正常血管化中的作用。我们假设视网膜的正常发育不会受到EC nachr的药理拮抗作用的不利影响。这一假设部分基于a7-nAChR缺陷小鼠的正常表型。我们将首先确定在正常发育过程中，nAChR通路关键元件的表达水平和定位，包括高亲和力胆碱转运蛋白、胆碱乙酰转移酶和nAChR亚基。我们将采用激光捕获显微解剖、实时RT-PCR、原位杂交和免疫组织化学的方法研究正常视网膜血管的EC。我们将仔细评估a7-nAChR缺陷小鼠视网膜中的血管和神经元发育。最后，我们将确定眼睛中nachr的药理学拮抗剂（比较非选择性和7- nachr选择性拮抗剂）是否会对正常的神经元或血管发育产生不利影响。2. 在小鼠ROP模型中，通过药理学和基因敲低EC nachr来确定该通路的过度激活是否有助于视网膜新生血管的形成。使用上述方法，我们将确定视网膜新生血管是否与nAChR通路中任何成分的视网膜表达增加有关。我们将确定非选择性（甲美胺）或7-优先（甲基莱卡乌碱）nAChR拮抗剂是否抑制视网膜新生血管。nAChR拮抗剂对视网膜神经元功能的影响将通过eeg评估。我们将在a7- nAChR敲除小鼠和对照组小鼠中诱导ROP，以确定通过a7- nAChR信号传导是否有助于视网膜新生血管的形成。我们将进行生化研究，以评估血浆和视网膜乙酰胆碱水平之间的关系，并将这些水平与视网膜血管内皮生长因子（VEGF）、视网膜血管性和通透性水平的变化联系起来。

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.