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Vascular diseases are among the most common causes of morbidity and mortality in the world. A remarkable feature of these disorders in the focal nature of their distribution. In fact, no single vascular disease affects every blood vessel in the body. An important goal in vascular biology is to delineate mechanisms of vascular bed specific pathology. One clue to these patterns is found within the endothelium. The endothelium is a highly malleable cell layer, constantly responding to changes within the extra-cellular environment and responding in ways that are usually beneficial, but at times harmful to the organism. Growth factors, cytokines, serine proteases and other mediators activate gene transcription in endothelial cells, resulting in multiple phenotypic changes, including hemostatic balance, permeability, leukocyte adhesion, migration and angiogenesis. Endothelial cell activation is not an all-or-none response. Indeed, different extra-cellular mediators engage the endothelium in ways that differ from one signal to the next. A major focus in my lab is to study the temporal and spatial dynamics of endothelial cell phenotypes.
Experimental design 1 Using DNA microarrays, we are carrying out a global survey of mRNA expression profiles in primary endothelial cells cultured in the absence or presence of various growth factors or cytokine. We have previously demonstrated that (1) thrombin induces the up-regulation and down-regulation of multiple genes in the endothelium (Fig. 1), (2) thrombin-mediated gene expression involves a multitude of transcription factors. We believe future breakthroughs in the field will depend on a better understanding of the spatial and temporal dynamics of these transcriptional networks.
Experimental design 2 While most of the gene expression studies in endothelial cells have been carried out in cell culture systems, a growing list of endothelial cell-specific promoters have also been shown to direct expression in the endothelium of transgenic mice. However, an important limitation of standard transgenic assays is that multiple copies of the transgene are inserted randomly into the mouse genome, resulting in line-to-line variation in expression. One way to control for these variables is to target a single copy of the transgene to a defined locus in the mouse genome by homologous recombination. In this lab, we have used such an approach to target several promoters, VWF, Flt-1, e-NOS and Tie-2, to the hypoxanthine phosphoribosyltransferase (Hprt) gene locus. Our results demonstrate that the VWF promoter contains information for specific gene expression in the heart and brain. The Flt-1 promoter directs expression in all vascular beds except for those in the liver. A small Tie-2 promoter fragment (723-bp promoter) directed widespread endothelial cell expression (Fig. 2). Using this system, we will not only uncover the nature of vascular heterogeneity, but also provide a useful tool to direct heterologous gene expression within the intact endothelium.
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Systems-Based Medical Depts
