The development of the vascular system is dependent on a tightly regulated program beginning with the differentiation of endothelial cells from mesoderm, followed by their subsequent assembly into vascular channels, and ending with the establishment of organ and region-specific endothelial cell properties. Phenotypic diversity, which allows the endothelium to carry out specialized tasks according to the needs of the local environment, has been described at the level of cellular structure, function and antigen composition. However, the molecular pathways involved in establishing and maintaining these phenotypic patterns remain poorly defined. As one approach to this problem, we have focused on the mechanisms that govern regional differences in the expression of the von Willebrand factor (vWF) gene. We have shown in transgenic mice that distinct promoter elements of the human vWF gene direct expression to different vascular beds. Specifically, we have identified sequences within the 5' region and/or first intron that contain information for expression either within the blood vessels of the brain or microvessels of the heart. In addition, we have shown that transgene activation is mediated by signals residing in the extracellular milieu. These results raise the interesting possibility that vWF and perhaps other endothelial cell genes are differentially regulated by region-and organ-specific signaling pathways. The long range goals of this proposal are to elucidate the molecular basis of differential expression of the vWF gene. In the first two aims, we will characterize the DNA:protein interactions that mediate heart microvascular bed-specific expression of vWF. To delineate the putative transcriptional control elements, we will use screening strategies which take into account the potential role of environmental signals in modulating endothelial cell gene expression. These will include a combination of transgenic, co-culture and in vivo liposome- based transient transfection assays. We will then use a lambdagt11 or yeast-based screening system to clone the transcription factor that interacts specifically with these sequences. In the third and final aim, we will characterize the heart endothelial cell-specific transcriptional pathway in non-expressing vascular beds. We will reconstitute the signaling pathway under in vitro and in vivo conditions and determine the extent to which the transcriptional network is inducible in non-cardiac endothelial cells. Taken together, these studies should provide important information about the mechanisms of endothelial cell heterogeneity and vascular diversity.
Showing the most recent 10 out of 18 publications
