Signal-transduction of Endothelial nAChR in Angiogenesis

描述(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因子[1]。我们的研究表明，内皮烟碱型乙酰胆碱受体(NAChR)介导了尼古丁的这种作用。虽然有大量的文献关于神经元nAChR的受体组成、分布和信号转导，但对最近发现的内皮烟碱胆碱能通路中的信号转导知之甚少。此外，目前还没有关于血管生成刺激(如缺氧)如何招募这一途径的信息。因此，我们的具体目标是：1)研究缺氧对nAChR表达的影响以及内皮细胞合成和释放ACh的细胞内机制，包括胆碱转运体、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亚基以及缺氧或血管内皮生长因子对它们的调节。我们将使用多种遗传学或药理学工具来下调nAChR途径中可能的元件的表达或活性，并观察其对钙离子通量、NO精加工和EC迁移的功能影响。2)描述内皮型nAChR激活的信号通路(S)，重点介绍那些已知参与NO合成酶激活的信号通路(因为NO似乎是nAChR诱导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Western分析、激酶活性测定，并观察特定的药理拮抗剂、显性负性突变体或RNAi对钙离子通量、NO精加工和EC迁移的影响。我们将利用消减抑制杂交和基因芯片技术寻找受S(NAChR)独有调控的基因，并借助SAM、GeneMAPP、层次聚类等分析工具进一步表征表达的时间过程、剂量反应和内皮细胞的选择性，将原始的微阵列数据转化为有用的信息。在已确定的受调控的、内皮选择性尼古丁特异基因中，最初最有希望的候选基因之一将成为旨在阐明该基因在nAChR血管生成效应中的作用的体外机制研究的目标(S)。

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).