Analysis of the VEGFR1 (FLT-1) Isoforms in Vascular Development

While sprouting angiogenesis is critical to blood vessel formation, the cellular and molecular controls of this process are poorly understood. As blood vessels form, the endothelial cells that comprise the vessel must coordinate the processes of cell division and cell migration for proper vascular morphogenesis to occur. Vascular endothelial growth factor-A (VEGF-A), an endothelial cell-specific ligand, is critical for these endothelial cell responses. Two high affinity receptors for VEGF-A, Flk-1 and Flt-1, are responsible for the effects of VEGF-A on vascular development. While VEGF-A signaling through Flk-1 has been shown to stimulate endothelial cell division, migration, and survival, the role of Flt-1 is less clear. Mice lacking the flt-1 receptor die around mid-gestation (E9.5) due to vascular overgrowth and disorganization. It is thought that Flt-1 acts to sequester VEGF-A, thereby inhibiting signaling through Flk-1. In my thesis work, I have used time-lapse imaging of GFP-expressing vessels derived from mouse embryonic stem (ES) cells, to reveal a role for flt-1 in modulating vascular branch (sprouts) formation and migration. Analysis of timelapse data also reveals that flt-1 mutant vessels have increased rates of endothelial cell division. This data supports a model whereby the role of flt-1 is to positively regulate sprout formation and migration and negatively regulate endothelial cell proliferation. Flt-1 is expressed as a full-length receptor tyrosine kinase (mFlt-1) and as a soluble isoform (sFlt-1). In order to get a clearer understanding of the role of the flt-1 isoforms in vascular development, I have reintroduced each flt-1 isoform into flt-1 mutant ES cells. My results reveal that the sFlt-1 isoform rescues the vessel branching morphogenesis defect observed in flt-1 mutant vessels, and that it does so more efficiently than the mFlt-1 isoform. Furthermore, both flt-1 isoforms appear to partially rescue the cell proliferation defect. These results support a model whereby Flt-1 primarily affects vascular branching morphogenesis via a soluble molecule, sFlt-1, perhaps by affecting the formation of a VEGF-A gradient that normally provides directional information for sprouts. These results also reveal that both Flt-1 isoforms have the ability to regulate endothelial cell proliferation