Signal-transduction of Endothelial nAChR in Angiogenesis

血管生成中内皮 nAChR 的信号转导

• 批准号: 6870383
• 负责人: JOHN P COOKE
• 金额: $ 24.81万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-04 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6870383
• 关键词: RNA interference; acetylcholine; acetylcholinesterase; angiogenesis; biological signal transduction; calcium flux; cell line; cell migration; choline acetyltransferase; clinical research; enzyme activity; flow cytometry; genetic regulation; high throughput technology; human genetic material tag; hypoxia; microarray technology; nicotinic receptors; nitric oxide; nitric oxide synthase; protein structure function; protein transport; receptor expression; subtraction hybridization; vascular endothelial growth factors; vascular endothelium; western blottings

DESCRIPTION (provided by applicant): Recently, we have made the novel and serendipitous observation that nicotine is an extraordinarily potent agent of angiogenesis [1]. Our studies indicate that endothelial nicotinic acetylcholine receptors (nAChR) mediates this effect of nicotine. Whereas there is a substantial body of literature regarding the receptor composition, distribution and signaling of neuronal nAChR, there is little known about signaling in the recently discovered endothelial nicotinic cholinergic pathway. Furthermore, there is no information about how this pathway is recruited by angiogenic stimuli (e.g. hypoxia). Accordingly, our specific aims are to: 1) Characterize the effect of hypoxia on the expression of nAChR and the intracellular machinery for synthesizing and releasing ACh from endothelial cells, including expression and activities of the choline transporter, choline acetyltransferase, and acetylcholinesterase. We will identify the nAChR subunits that are expressed on endothelial cells and their modulation by hypoxia or VEGF. We will use a variety of genetic or pharmacological tools to knock down the expression or activities of putative elements in the nAChR pathway, and observe the functional effect on calcium flux, NO elaboration and EC migration. 2) Characterize the signaling pathways activated by stimulation of endothelial nAChR(s), focusing on those that are also known to be involved in NO synthase activation (as NO appears to be a critical mediator of nAChR-induced angiogenesis). To dissect out the role of various signaling proteins, we will use Western analysis, kinase activity assays and observe the effect of specific pharmacological antagonists, dominant negative mutants or RNAi on calcium flux, NO elaboration and EC migration. We will identify genes uniquely regulated by nAChR(s) using subtraction suppression hybridization and DNA microarray, and further characterize the time course, dose response, and endothelial selectivity of expression, with the assistance of analytical tools including SAM, GeneMAPP, hierarchical clustering to transform the raw microarray data into useful information. Of the regulated, endothelial-selective nicotine-specific genes that are identified, initially one of the most promising candidates will become the target of in vitro mechanistic studies designed to elucidate the role of the gene in the angiogenic effects of the nAChR(s).

描述（由申请人提供）：最近，我们偶然发现尼古丁是一种非常有效的血管生成剂[1]。我们的研究表明内皮烟碱乙酰胆碱受体（nAChR）介导尼古丁的这种作用。尽管有大量关于神经元 nAChR 受体组成、分布和信号传导的文献，但对最近发现的内皮烟碱胆碱能通路中的信号传导却知之甚少。此外，没有关于血管生成刺激（例如缺氧）如何募集该通路的信息。因此，我们的具体目标是：1）表征缺氧对 nAChR 表达以及内皮细胞合成和释放 ACh 的细胞内机制的影响，包括胆碱转运蛋白、胆碱乙酰转移酶和乙酰胆碱酯酶的表达和活性。我们将鉴定在内皮细胞上表达的 nAChR 亚基及其通过缺氧或 VEGF 的调节。我们将使用各种遗传或药理学工具来敲低 nAChR 通路中推定元件的表达或活性，并观察对钙流动、NO 合成和 EC 迁移的功能影响。 2) 表征通过刺激内皮 nAChR 激活的信号通路，重点关注那些已知参与 NO 合酶激活的信号通路（因为 NO 似乎是 nAChR 诱导的血管生成的关键介质）。为了剖析各种信号蛋白的作用，我们将使用蛋白质印迹分析、激酶活性测定，并观察特定药理学拮抗剂、显性失活突变体或 RNAi 对钙通量、NO 合成和 EC 迁移的影响。我们将利用减法抑制杂交和DNA微阵列识别nAChR独特调控的基因，并借助SAM、GeneMAPP、层次聚类等分析工具进一步表征表达的时程、剂量反应和内皮选择性，将原始微阵列数据转化为有用的信息。在已鉴定的受调节的内皮选择性尼古丁特异性基因中，最初最有希望的候选基因之一将成为体外机制研究的目标，旨在阐明该基因在 nAChR 的血管生成作用中的作用。