Untersuchungen zur Funktion endothelialer Transkriptionsfaktoren bei der Angiogenese

Using the Cre-loxP recombination system it is possible to inactivate genes in a cell-type specific manner. In this study, regulatory elements of the flk-1 gene were fused to the Cre recombinase gene and transgenic mouse lines were established (flk-1-Cre). These mice were crossed to the reporter mouse line Rosa26R in order to analyse Cre activity in double transgenic offspring by LacZ staining. The analysis of flk-1-Cre/Rosa26R embryos or adult animals revealed a specific staining of endothelial cells of blood vessels in most organ systems. Moreover, Cre expression was detected in blood vessels of experimental BFS-1 tumours. Flk-1-Cre mouse lines can be used to inactivate floxed target genes specifically in endothelial cells. The role of hypoxia-inducible factors (HIF) during embryonic vessel development was analysed by blocking HIF signalling in endothelial cells. For that purpose, a dominant negative mutant of HIF2-alpha (HIFdn) was expressed in the endothelium of transgenic mouse embryos by using the gene regulatory elements of flk-1. HIFdn transgenic mouse embryos were growth retarded and died at day 11.5 of gestation. Primitive vascular networks were established, which indicated that in these transgenic embryos vasculogenesis was not impaired. But immature vessels were not remodelled as no angiogenic sprouting occurred. In hearts of transgenic embryos defects were observed in heart-looping and formation of the trabeculae. Gene expression analysis revealed a marked reduction of the expression levels of tie-2, flt-1 and flk-1. These results show that endothelial HIFs are essential for embryonic angiogenesis. The regulation of the human VEGF receptor-2 gene (KDR) was investigated. In reporter gene assays, the transcription factor HIF2-alpha, but not its relative HIF1-alpha, stimulated KDR promoter activity. Potential HIF2 binding sites were identified within the KDR promoter. Activation of the KDR promoter was abolished by mutating one of these sites. A region of high sequence similarity was found by comparing the first intron of the human and the mouse KDR gene. This region (enhancer) contained binding sites for transcription factors which were previously shown to be essential for endothelial cell-specific gene expression in vivo. These binding sites (SCL/Tal-1, Gata, Ets) were conserved in the first intron of the human gene, which implies a functional role in regulating gene expression.