Nicotinic receptor effect of ACh on skin endothelium

描述(申请人提供)：烟碱型乙酰胆碱受体(NAChRs)广泛表达于神经性和非神经性组织。在非神经性组织中，nAChRs参与伤口愈合、细胞增殖和血管生成。虽然已经在非神经元组织中发现了一些nAChR亚基，但结合形成功能性nAChRs的亚基组合尚未确定。此外，以前在血管生成方面研究nAChRs的实验依赖于不区分不同受体亚型的激动剂和拮抗剂。我们假设ACh与nAChRs结合激活了细胞内的信号通路，从而触发了血管生成。这可能与儿童皮肤血管畸形有关。因此，本研究的目的是研究nAChRs在血管生成中的整体作用，确定 具体受体亚型的贡献，并最终了解nAChRs激活影响血管生成的机制。其具体目的是：1)确定胆碱能激动剂和拮抗剂对正常组织新生血管生成的影响。这将通过体外血管外植体系统中的微血管萌发来评估。2)分析正常血管内皮细胞和真皮微血管内皮细胞亚基的表达。Subaim 1.RT-PCR研究将用于确定nAChR亚单位在正常小鼠血管内皮细胞、人血管内皮细胞(HUVEC)体外模型和人真皮来源微血管内皮细胞(HDMEC)中的表达模式。Subaim 2.确定受体 正常血管组织中表达的nAChRs的组成。虽然A3、A7和A9nAChR亚基已经在人主动脉内皮细胞中被鉴定，但它们在HUVEC和HDMEC上的表达以及其他亚基的表达还没有确定，组成功能性nAChRs的亚基的组合还有待确定。3)观察胆碱能激动剂对人脐静脉内皮细胞和人脐静脉内皮细胞CD40、VAMC-1和E-选择素表达的影响。这一结果可能对皮肤增生性血管病变(如血管瘤)和炎性血管病变(如中小血管炎)的发病机制和处理具有重要意义。

DESCRIPTION (provided by applicant): Nicotinic acetylcholine receptors (nAChRs) are widely expressed in neuronal and non-neuronal tissues. In non-neuronal tissue, nAChRs have been implicated in wound healing, cell proliferation and angiogenesis. Although a few nAChR subunits have been identified in non-neuronal tissues, the combinations of subunits that combine to form functional nAChRs have yet to be determined. In addition, previous experiments that addressed nAChRs in the context of angiogenesis relied upon agonists and antagonists that do not discriminate between different receptor subtypes. We hypothesize that the ACh binding to nAChRs activates intracellular signaling pathways that can trigger angiogenesis. This could have relevance to cutaneous vascular malformations that occur in children. Thus, the goals of this research are to investigate the overall role of nAChRs in angiogenesis, to identify the contributions of specific receptor subtypes, and ultimately to understand the mechanisms whereby activation of nAChRs affects angiogenesis. The Specific Aims are: 1) To determine the effects of cholinergic agonists and antagonists on de novo angiogenesis in normal tissue. This will be evaluated by microvascular sprouting in an ex vivo vascular explant system. 2) To analyze subunit expression in normal vascular endothelial cells and in dermal microvascular endothelial cells. Subaim 1. RT-PCR studies that rely on tested primers sets will be used to determine the expression pattern of nAChR subunits in vascular endothelial cells from normal mice, in the human vascular endothelial cell (HUVEC) in vitro model, and in the human dermal derived microvascular endothelial cells (HDMEC). Subaim 2. To determine the receptor composition of nAChRs expressed in normal vascular tissue. Although a3, a7, and a9 nAChR subunits have been identified in human aortic endothelial cells, their expression on HUVEC and HDMEC as well as the expression of other subunits have not yet been identified and the combinations of subunits that comprise functional nAChRs have yet to be determined. 3) Finally we will evaluate changes in the expression of CD40, VAMC-1, and E-selectin by HUVECs and HDMECs following the administration of cholinergic agonists. The results could have important implications for the pathogenesis and management of proliferative vascular lesions that occur in the skin such as hemangiomas and in inflammatory vascular lesions such as small and medium vessel vasculitis.