Signal-transduction of Endothelial nAChR in Angiogenesis

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。虽然有大量的文献关于神经元nAChR的受体组成，分布和信号传导，但对最近发现的内皮烟碱胆碱能通路中的信号传导知之甚少。此外，没有关于该途径如何被血管生成刺激（例如缺氧）募集的信息。因此，我们的具体目标是：1）表征缺氧对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。我们将确定的nAChR亚单位，表达内皮细胞和缺氧或VEGF的调制。我们将使用各种遗传学或药理学工具敲低nAChR通路中推定元件的表达或活性，并观察对钙通量、NO产生和EC迁移的功能效应。2)表征通过刺激内皮nAChR激活的信号传导途径，重点关注已知参与NO合酶激活的信号传导途径（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Western分析，激酶活性测定，并观察特定的药理学拮抗剂，显性负突变体或RNAi对钙流，NO加工和EC迁移的影响。我们将使用减法抑制杂交和DNA微阵列来鉴定由nAChR（s）独特调控的基因，并进一步表征表达的时间过程、剂量反应和内皮选择性，在分析工具包括SAM、GeneMAPP、分层聚类的帮助下，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血管生成作用中的作用。

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).