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，我们做出了新颖和偶然的观察，即尼古丁是一种非常有效的血管生成药物[1]。我们的研究表明，内皮烟碱乙酰胆碱受体（NACHR）介导了尼古丁的这种作用。尽管神经元NACHR的受体组成，分布和信号传导有大量文献，但在最近发现的内皮烟碱胆碱能途径中，信号传导鲜为人知。此外，没有关于该途径如何通过血管生成刺激募集的信息（例如缺氧）。因此，我们的具体目的是：1）表征缺氧对NACHR表达和细胞内机械表达的影响，用于从内皮细胞中合成和释放ACH，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。我们将确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工具来击倒NACHR途径中推定元素的表达或活动，并观察到对钙通量的功能影响，没有阐述和EC迁移。 2）表征通过刺激内皮NACHR激活的信号传导途径，重点是已知参与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体）。为了剖析各种信号蛋白的作用，我们将使用西方分析，激酶活性测定，并观察特定的药理学拮抗剂，显性阴性突变体或RNAi对钙通量，没有阐述和EC迁移的影响。我们将使用减法抑制杂交和DNA微阵列确定由NACHR唯一调节的基因，并在分析工具的帮助下，进一步表征了时间过程，剂量响应和表达的内皮选择性，包​​括SAM，Genemapp，GeneMapp，Genemapp，层次结构，层次聚类以将原始微阵列数据转换为有用的信息。在被鉴定出的受调节的内皮选择性尼古丁特异性基因中，最初是最有前途的候选者之一将成为旨在阐明基因在NACHR血管生成作用中的作用的体外机械研究的靶标。