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。我们的研究表明，内皮尼古丁乙酰胆碱受体（nAChR）介导尼古丁的这种作用。虽然关于神经元nAChR的受体组成、分布和信号传导的文献大量，但最近发现的内皮尼古丁胆碱能通路的信号传导却知之甚少。此外，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)表征缺氧对内皮细胞合成和释放乙酰胆碱的nAChR表达和细胞内机制的影响，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亚基及其受缺氧或VEGF的调节。我们将使用多种遗传或药理学工具来敲低nAChR通路中假定元件的表达或活性，并观察其对钙通量、NO细化和EC迁移的功能影响。2)表征内皮细胞nAChR刺激激活的信号通路，重点关注那些已知参与NO合成酶激活的信号通路（因为NO似乎是nAChR诱导的血管生成的关键介质）。为了分析各种信号蛋白的作用，我们将使用Western分析、激酶活性分析，并观察特定药理拮抗剂、显性阴性突变体或RNAi对钙通量、NO细化和EC迁移的影响。我们将使用减法抑制杂交和DNA芯片识别nAChR(s)唯一调控的基因，并进一步表征时间过程、剂量反应和内皮表达选择性，借助分析工具，包括SAM、GeneMAPP、分层聚类，将原始芯片数据转化为有用的信息。在已确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).