The Role of the Nicotinic Cholinergic Pathway in Retinopathy of Prematurity

烟碱胆碱能通路在早产儿视网膜病变中的作用

• 批准号: 8529537
• 负责人: JOHN P COOKE
• 金额: $ 38.11万
• 依托单位: METHODIST HOSPITAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8529537
• 关键词: Acetylcholine; Affect; Angiogenic Factor; Arterial Fatty Streak; Biochemical; Biological; Birth; Blindness; Blood Vessels; Child; Choline O-Acetyltransferase; Cholinergic Receptors; Choroid; Choroidal Neovascularization; Development; Disease; Drug Formulations; Elements; Endothelial Cells; Exudative age-related macular degeneration; Eye; Genetic; Growth Factor; Human Volunteers; Hypoxia; Immunohistochemistry; Impairment; In Situ Hybridization; Infant; Inflammatory; Ischemia; Knockout Mice; Lead; Learning; Ligands; Malignant neoplasm of lung; Mecamylamine; Mediating; Medicine; Methods; Modeling; Mus; Nature; Neurons; Nicotinic Receptors; Pathogenesis; Pathologic Neovascularization; Pathway interactions; Patients; Permeability; Phase; Phase II Clinical Trials; Phenotype; Plasma; Pregnancy; Premature Infant; Receptor Activation; Research; Retina; Retinal; Retinal Detachment; Retinal Diseases; Retinal Neovascularization; Retinopathy of Prematurity; Reverse Transcriptase Polymerase Chain Reaction; Risk Factors; Role; Safety; Sclera; Signal Transduction; Signaling Molecule; Stimulation of Cell Proliferation; Time; Traction; Tube; Tumor Angiogenesis; United States; Vascular Endothelial Growth Factors; Vascularization; Vision; angiogenesis; base; cancer cell; choline transporter; cholinergic; diabetic; human subject; insight; laser capture microdissection; macular edema; methyllycaconitine; migration; neovascularization; neuron development; novel; novel strategies; novel therapeutic intervention; novel therapeutics; phase 1 study; prevent; receptor; retina blood vessel structure; retinal neuron; tumor growth

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 ¿7-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 ¿7-nAChR deficient mouse. Finally, we will determine if pharmacological antagonism of nAChRs in the eye (comparing non-selective versus ¿7-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 ¿7-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 ¿7- nAChR knockout mice and littermate controls to determine if signaling through ¿7- 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.

早产儿视网膜病变(ROP)是导致儿童视力受损和失明的主要原因。 美国，由于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 这与病理性新生血管有关。这一途径是由内皮细胞烟碱介导的 乙酰胆碱受体(NAChR)。内皮细胞nAChR是一种配体门控的阳离子通道，由 内源性信号分子乙酰胆碱(ACh)。该受体的激活可诱导内皮细胞 有丝分裂、迁移和管状形成，促进血管生成。该途径被上调为 低氧，以及其他血管生成因子，如血管内皮生长因子(VEGF)。因此， 我们提出以下具体目标，以开发一种治疗ROP的新方法： 1.鉴定发育中视网膜内皮细胞烟碱胆碱能途径，并测定其 在正常血管形成中的作用。我们假设视网膜的正常发育不会对 受EC nAChRs药理拮抗作用的影响。这一假设在一定程度上是基于常态 7-nAChR缺陷小鼠的表型。我们将首先确定，在正常发展过程中， 包括高亲和力胆碱在内的nAChR途径关键元件的表达水平和定位 转运蛋白、胆碱乙酰转移酶和nAChR亚基。我们将研究正常视网膜血管中的内皮细胞 采用激光捕获显微切割和实时RT-PCR、原位杂交和免疫组织化学方法。 我们将仔细评估7-nAChR缺陷小鼠视网膜中血管和神经元的发育。 最后，我们将确定nAChRs在眼睛中的药理拮抗作用(与非选择性比较 与7-nAChR选择性拮抗剂相比)将对正常的神经元或血管发育产生不利影响。 2.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 利用EC nAChRs的药理学和基因敲除建立ROP小鼠模型。使用 方法如上所述，我们将确定视网膜新生血管是否与视网膜增厚相关 NAChR途径的任何组成部分的表达。我们将确定是否对非选择性的 (甲戊胺)或7-优先(甲基乌头碱)nAChR拮抗剂抑制视网膜 新生血管。NAChR拮抗剂对视网膜神经功能的影响将通过ERGs进行评估。 将在7-nAChR基因敲除小鼠和产仔对照小鼠中诱导ROP，以确定通过7-nAChR基因敲除的小鼠和产仔对照组的信号是否 NAChRs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ACh水平之间的关系，并将这些水平与视网膜中ACh水平的变化相关 血管内皮生长因子(VEGF)、视网膜血管通透性。