Abstract 14: The Helix-Loop-Helix Transcription Factor nPAS4 Induces Sprouting Angiogenesis and Regulates Tip Cell Formation

Introduction: Neuronal PAS4 (nPAS4) -formerly known as LE-PAS- is a helix-loop-helix-PAS (HLH-PAS) transcription factor that has recently been cloned as the vertebrate homologue of the drosophila protein dysfusion ( dys ). In drosophila, the lack of dys is characterized by impaired midline fusion of tracheal tubes. Forced expression of dys results in aberrant sprouting of tracheal tubes in drosophila. In neurons, nPAS4 plays a role in the formation of GABA-releasing synapses. Based on the morphological similarity between tracheal tube formation and angiogenesis, we aimed to investigate the role of nPAS4 in angiogenesis in vertebrates. Methods and Results: RT-PCR and Western blotting analyses showed that nPAS4 is expressed at low levels in various endothelial cell lines. The expression of nPAS4 is not upregulated by angiogenic growth factors but is induced by hypoxia. Moreover, membrane depolarization using potassium chloride results in a calcium-dependent upregulation of nPAS4. When nPAS4 expression is blocked via siRNA, endothelial cell sprouting is significantly reduced. In contrast, overexpression of nPAS4 in endothelial cells results in enhanced sprouting and branching in two- and three-dimensional models. Indeed the formation of tip cells is dependent on the presence of nPAS4 in the endothelial cell spheroid sprouting model. Accordingly, when nPAS4 is overexpressed, tip cell formation is strongly enhanced. Furthermore, the number of filopodia -a typical feature of tip cells- is increased in nPAS4 overexpressing cells, as visualized by actin staining in immunocytochemistry. In a Transwell Migration Assay, endothelial cell migration was found to be inhibited by nPAS4. Further experiments traced this back to a stronger adhesion of the cells to different extracellular matrices. Using fli-GFP transgenic zebrafish as an in vivo model, we found that a knock-down of nPAS4 causes disturbed intersomitic blood vessel formation. Conclusions: NPAS4 is a novel HLH-PAS transcription factor that regulates endothelial tip cell formation and endothelial cell sprouting.