Abstract 5647: Impact of Growth Factors, Shear Stress and Transcription Factor COUP-TFII on the Notch Pathway in Human Endothelial Cells

Abstract

Arterial and venous endothelial cells can differ in vascular function thus leading to vessel-specific diseases. The molecular mechanisms for differentiation and maintenance of the phenotype of arterial and venous endothelial cells are not well understood. Using primary cultures of human umbilical artery (HUAEC) or vein (HUVEC) endothelial cells we identified by DNA microarrays Hey2 as arterial and COUP-TFII as venous marker gene. Differential expression of these genes and preferential arterial expression of further genes of the Notch signalling pathway (Dll4, Notch4, Hey1, Hey2) were confirmed by RT- and real-time PCR in independent HUAEC and HUVEC preparations. Differential protein expression of Hey2 and COUP-TFII was confirmed as well. We further showed exclusive expression of Hey2 and COUP-TFII in HUAEC and HUVEC over several passages (up to passage 12). Next, we analyzed the impact of growth factors on the preferential arterial genes of the Notch pathway. In medium supplemented with different growth factors (IGF-1, hEGF-1, PDGF-BB, bFGF-2 or VEGF-A) only VEGF-A was able to induce expression of Dll4, Notch4 and Hey1 in venous endothelial cells. A further increase of the high arterial Dll-4 and Hey1 expression was achieved by VEGF-A incubation in HUAEC. This increased expression of Dll-4 and Hey-1 was prevented by VEGF receptor-2 (KDR) inhibitor SU5416. Inhibition of the Notch pathway by gamma-secretase inhibitor X (GSI-X) reduced Dll-4 and Hey-1 expression in venous and arterial endothelial cells without effecting Hey2 expression. In order to reduce arterial Hey2 expression we next applied shear stress (1 dyn/cm 2 or 30 dyn/cm 2 ) to arterial endothelial cells. The main arterial transcription factor Hey2 was not changed. Next, we cloned full-length and terminal deletions of the human Hey2 promoter (−2132/+194; −1080/+194; −418/+194; −54/+194 bp). All fragments showed high basal activity (82-, 84-, 293-, 25-fold). Finally, double transfection with a COUP-TFII overexpression plasmid reduced Hey2 promoter activity of the −2132/+194 fragment by 50%. In conclusion, our data suggest an interaction of the venous marker COUP-TFII and the arterial marker Hey2 at the Hey2 promoter.