A better understanding of the process of blood vessel formation and stabilization is important during development and in many disease states such as coronary artery disease, tumor progression or diabetes. The mechanism how vascular progenitor cells differentiate into arterial or venous endothelial cell fate is still under debate. We and others have identified marker genes which are preferentially expressed in arterial (e.g. Dll4, Notch4, Hey1 and Hey2) or venous (COUP-TFII) endothelial cells. We were especially interested in the arterial transcription factor Hey2 and the venous transcription factor COUP-TFII.

The aim of this study was to convert human venous endothelial cells into an arterial phenotype and vice versa. Due to low transfection efficiency of primary endothelial cells by all tested lipid based transfection reagents (e.g. TransFectin, Lipofectamine, FuGeneHD) we established lentiviral gene transfer for overexpression of Hey2 and COUP-TFII in arterial and venous endothelial cells. In order to quantify transduction efficiency, first human arterial and venous endothelial cells were infected with a GFP lentivirus and analysed after 24 hours by FACS. Using this approach we achieved very high transduction efficiency in arterial (up to 70%) and venous endothelial cells (up to 96%). Because the lentiviral vector has the ability to integrate the target gene into the host genome, we further analysed the GFP expression in higher cell passages. We transduced arterial and venous endothelial cells with the GFP lentivirus, cultured the cells for several passages and monitored GFP expression by immunofluorescence microscopy. Even after four passages, about 90% of the cells were GFP positive. Next, we transduced arterial endothelial cells with COUP-TFII lentiviruses and venous endothelial cells with Hey2 lentiviruses. After 24, 48 and 72 hours, protein expression was analysed by Western blot. We achieved high levels of COUP-TFII protein expression in arterial cells and high Hey2 protein expression in venous endothelial cells (normalized to GAPDH). The expression reached even the level of basal expression of endogenous COUP-TFII or Hey2 in the corresponding other cell type. COUP-TFII is not only highly expressed in transduced arterial endothelial cells, but further processed and translocated into the nucleus. Overexpression of COUP-TFII in arterial endothelial cells leads to a downregulation of the arterial transcription factor Hey2.

In conclusion, we have established a safe, efficient and long-lasting lentiviral gene transfer for COUP-TFII and Hey2 in primary human endothelial cells. Using this approach overexpression of COUP-TFII may lead to a conversion of endothelial cells from an arterial into a venous phenotype.