The mechanisms involved in the regulation of vasculogenesis still remain unclear in mammals. Totipotent embryonic stem (ES) cells may represent a suitable in vitro model to study molecular events involved in vascular development. In this study, we followed the expression kinetics of a relatively large set of endothelial-specific markers in ES-derived embryoid bodies (EBs). Results of both reverse transcription-polymerase chain reaction and/or immunofluorescence analysis show that a spontaneous endothelial differentiation occurs during EBs development. ES-derived endothelial cells express a full range of cell lineage-specific markers: platelet endothelial cell adhesion molecule (PECAM), Flk-1, tie-1, tie-2, vascular endothelial (VE) cadherin, MECA-32, and MEC-14.7. Analysis of the kinetics of endothelial marker expression allows the distinction of successive maturation steps. Flk-1 was the first to be detected; its mRNA is apparent from day 3 of differentiation. PECAM and tie-2 mRNAs were found to be expressed only from day 4, whereas VE-cadherin and tie-1 mRNAs cannot be detected before day 5. Immunofluorescence stainings of EBs with antibodies directed against Flk-1, PECAM, VE-cadherin, MECA-32, and MEC-14.7 confirmed that the expression of these antigens occurs at different steps of endothelial cell differentiation. The addition of an angiogenic growth factor mixture including erythropoietin, interleukin-6, fibroblast growth factor 2, and vascular endothelial growth factor in the EB culture medium significantly increased the development of primitive vascular-like structures within EBs. These results indicate that this in vitro system contains a large part of the endothelial cell differentiation program and constitutes a suitable model to study the molecular mechanisms involved in vasculogenesis.

Vascular endothelial cadherin (VE cadherin) gene encodes a Ca2؉ -dependent cell adhesion molecule required for the organization of interendothelial junctions. This gene is exclusively and constitutively expressed in endothelial cells. Previous data with transgenic mice revealed that the transcriptional regulatory elements present within a ؊2486/؉24 DNA fragment of mouse VE cadherin gene mimic the tissue-specific activity of the endogenous promoter. In this study, we analyzed elements implicated in the function of the proximal regulatory region. Electrophoretic mobility shift assay identified a GT-rich sequence (positions ؊49/؊39) interacting with factors related to the Sp1 family. Point mutations abolished the binding of nuclear proteins in vitro and drastically diminished the activity of the promoter in transient transfection assay. Supershift assays with antibodies against proteins of the Sp1 family revealed that Sp1 and Sp3 interact with this region of the VE cadherin promoter. Furthermore, two GGAA motifs, located at positions ؊93/؊90 and ؊109/؊106, were shown to interact with nuclear factors. Site-directed mutagenesis of these sequences demonstrated that these Ets binding sites are essential for promoter activity. In vitro binding assays in the presence of various antisera suggest that Erg is one of the proteins interacting with the ؊109/؊106 site.