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.
Sub aim 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).
Sub aim 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.
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