It was determined that the endothelial cell-specific cytokine vascular endothelial growth factor (VEGF) increased the level of ETS1 mRNA by two- to four fold. We have also determined that ETS1 protein level in VEGF-treated endothelial cells was increased. These observations have been made in both human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells (HMVEC-L). The response of these cells to VEGF occurs within 4 hours of treatment and occurs at an optimal concentration of 10-20 ng/ml VEGF. In order to determine a direct role for ETS1 in angiogenesis-associated endothelial cell phenotype, cells were treated with antisense oligonucleotides using lipofectin as a carrier. After treatment, the cells were assayed for their ability to migrate through matrigel-coated membranes. We have found that antisense ETS1 oligonucleotides inhibits this migration up to 75%. Sense or random sequence oligonucleotides had no effect. There was only a slight reduction in the level of ETS1 mRNA in the antisense-treated cells. However, antisense oligonucleotides lowered the level of u-Pa mRNA. For immortalization, HUVEC and HMVEC-L have been infected by Adeno12-SV40 hybrid virus (Ad12-SV40), amphotropic retroviruses expressing HPV-16 E6 and E7, or transfected with the HPV-18 genome. They expressed Factor VIII-related antigen and integrin alpha/v/beta3, take up fluorescent-acetylated low density lipoprotein (Dil-Ac-LDL), and develop capillary-like tube structures upon reaching confluence, indicating that the immortalized cells retain endothelial cell-specific features.
