The Proangiogenic Effect of Iroquois Homeobox Transcription Factor Irx3 in Human Microvascular Endothelial Cells

Angiogenesis is a dynamic process required for embryonic development. However, postnatal vascular growth is characteristic of multiple disease states. Despite insights into the multistep process in which adhesion molecules, extracellular matrix proteins, growth factors, and their receptors work in concert to form new vessels from the preexisting vasculature, there remains a lack of insight of the nuclear transcriptional mechanisms that occur within endothelial cells (ECs) in response to VEGF. Iroquois homeobox gene 3 (Irx3) is a transcription factor of the Iroquois family of homeobox genes. Irx homeodomain transcription factors are involved in the patterning and development of several tissues. Irx3 is known for its role during embryogenesis in multiple organisms. However, the expression and function of Irx3 in human postnatal vasculature remains to be investigated. Here we show that Irx3 is expressed in human microvascular endothelial cells, and expression is elevated by VEGF stimulation. Genetic Irx3 gain and loss of function studies in human microvascular endothelial cells resulted in the modulation of EC migration during wound healing, chemotaxis and invasion, and tubulogenesis. Additionally, we observed increased delta-like ligand 4 (Dll4) expression, which suggests an increase in EC tip cell population. Finally, siRNA screening studies revealed that transient knockdown of Hey1, a downstream Notch signaling mediator, resulted in increased Irx3 expression in response to VEGF treatment. Strategies to pharmacologically regulate Irx3 function in adult endothelial cells may provide new therapies for angiogenesis.