Endothelial cell phenotypes display remarkable heterogeneity in health and disease. An important goal in vascular biology is to understand the molecular mechanisms underlying the spatial and temporal modulation of endothelial cell phenotypes. One approach to this problem is to delineate mechanisms of endothelial cell gene regulation. We have recently proposed a model of endothelial cell-specific gene expression that emphasizes the importance of vascular bed-specific signaling pathways. According to this hypothesis, the expression of a single gene within the endothelium is regulated not by a common transcriptional control mechanism (or master switch), but rather by a constellation of site-specific pathways that begin in the extracellular environment and end at distinct regions of the promoter. The overall goal of this proposal is to map basal and inducible site-specific transcriptional modules in different organs (lung and brain) and different segments of the vascular tree (arteries and veins). This information will then be used to: 1) probe for mechanisms of endothelial cell heterogeneity, and 2) generate synthetic promoters for purposes of optimizing spatial and temporal control of gene expression. The primary focus of the first specific aim will be to delineate site-specific DNA elements of the human von Willebrand factor, Fit-1 and Tie-2 promoters. In addition, fate-mapping studies will be employed to track the longitudinal history of transgene expression over the lifetime of the animal. Two subsidiary studies will be carried out that address important questions relating to the interpretation of the results of transgenic assays and Hprt targeting, namely the degree to which the choice of reporter gene contributes to microheterogeneity, and the role ofgenetic background in determining the level and pattern of transgene expression, in the second specific aim, Hprt-targeted mice will be generated with synthetic endothelial cell-specific promoters that carry information for spatially- or temporally-restricted gene expression under normal conditions and in response to pathophysiological stimuli, including hypoxia and sepsis.
The third aim will evaluate the capacity of synthetic promoters to achieve site-specific specific expression in the context of adenoviral gene transfer vectors. Together, these studies will provide new insights into the modular control of endothelial cell gene expression and in doing so facilitate the development of selective gene targeting strategies in cardiovascular disease.
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