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）是美国儿童视力受损和失明的主要原因，原因是病理性视网膜新生血管形成。我们已经发现了一种新的血管生成途径，参与病理性新生血管形成。该途径由内皮烟碱乙酰胆碱受体（nAChR）介导。内皮nAChR是一种配体门控阳离子通道，由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、迁移和管形成并促进血管生成。该途径被缺氧和其他血管生成因子如血管内皮生长因子（VEGF）上调。因此，我们提出以下具体目标来开发用于ROP的新疗法：1.表征视网膜发育中的内皮烟碱胆碱能通路，并确定其在正常血管形成中的作用。我们推测，视网膜的正常发育不会受到EC nAChRs的药理学拮抗作用的不利影响。该假设部分基于α 7-nAChR缺陷小鼠的正常表型。我们将开始确定，在正常发育过程中，表达水平和nAChR途径的关键要素，包括高亲和力胆碱转运蛋白，胆碱乙酰转移酶，和nAChR亚基的本地化。我们将采用激光捕获显微切割技术、真实的实时荧光定量PCR、原位杂交和免疫组织化学等方法对正常视网膜血管内皮细胞进行研究。我们将仔细评估α 7-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的药理学拮抗作用（比较非选择性与α 7-nAChR选择性拮抗剂）是否会对正常神经元或血管发育产生不利影响。2.使用EC nAChR的药理学和遗传敲低，确定该途径的过度激活是否有助于ROP小鼠模型中的视网膜新生血管形成。使用上述方法，我们将确定视网膜新生血管是否与nAChR通路任何组分的视网膜表达增加相关。我们将确定非选择性（美加明）或α 7-优先（甲基利卡尼汀）nAChR拮抗剂的给药是否抑制视网膜新生血管形成。将通过ERG评估nAChR拮抗剂对视网膜神经元功能的影响。将在α 7- nAChR敲除小鼠和同窝对照中诱导ROP，以确定通过α 7-nAChR的信号传导是否有助于视网膜新生血管形成。我们将进行生化研究，以评估血浆和视网膜ACh水平之间的关系，并将这些水平与血管内皮生长因子（VEGF），视网膜血管分布和渗透性的视网膜水平的变化相关联。 公共卫生相关性：异常血管形成是早产儿视网膜病变的危险因素。早产儿视网膜病变（ROP）是超过50%的早产儿由于视网膜中异常血管形成而导致视力受损和失明的重要原因。ROP的可能危险因素是这些婴儿出生后的氧合和导致异常血管形成的生长因子的积累。在这项研究中，我们打算了解一种特殊受体在这些异常血管中的作用，并开发一种新的治疗方法来预防早产儿的视力丧失和失明。

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.