Abstract C38: Iroquois homeobox gene 3 is temporally regulated in response to vascular endothelial growth factor and modulates endothelial cell migration and tubulogenesis
Authors: Scarlett Kisha A. , Pattabiraman Vaishnavi , Anderson Leonard M. 
Published: August 2014

Abstract
Introduction: Differences in tumor-induced angiogenesis contributes to cancer-related health disparities. Understanding the transcriptional regulation of angiogenesis within endothelial cells (ECs) is essential to the development of anti-angiogenic therapies. Here we report that the homeobox gene, Irx3 is expressed in human microvascular endothelial cells (HMECs) and modulates their migration and ability to form networks during angiogenesis.
Methods: To test the effects of vascular endothelial growth factor (VEGF) on Irx3 expression, HMECs were treated with VEGF for 0 to 48 hours followed by extraction of total RNA and protein. Irx3 mRNA and protein expression were determined using qRT-PCR and western immunoblot respectively. Wound-healing and transwell migration assays were performed to investigate the role of Irx3 on EC migration in response to VEGF. For migration experiments, HMECs were transduced with adenoviral vectors resulting in the loss-of (Ad.mirIrx3-GFP) or gain-of (Ad.Irx3-V5) Irx3 function. We conducted a 672 transcription factor (TF) siRNA screen to identify transcriptional regulators of Irx3 expression in response to VEGF. HMECs were reverse-transfected with siRNAs and treated with VEGF for 12 hours. Total RNA was extracted for RT-PCR to determine Irx3 expression. Finally, we performed in vitro endothelial cell tubulogenesis assays using HMECs transduced with Ad.Irx3-V5, Ad.mirIrx3-GFP and their respective controls to study changes in endothelial tube formation as a result of differential Irx3 expression.
Results: VEGF treatment increases Irx3 expression at the mRNA and protein levels, with peak expression at 12 hours. HMECs transduced with Ad.mirIrx3-GFP exhibited decreased migration at 12 hours compared to HMECs transduced with Ad.Irx3-V5. Furthermore, when HMECs are transduced with Ad.Irx3-V5, they exhibit increased migration and invasiveness in transwell migration assays and form more complex networks during tube formation. The siRNA screen suggests that Irx3 is regulated by HEY1, a known transcriptional regulator of angiogenesis.
Conclusion: Our results provide compelling evidence that the homeobox gene Irx3 regulates EC migration, and is directly regulated by TFs known to govern transcriptional signaling during angiogenesis.
Grant Support: This work was supported in part by the following grants; the CVRI Center of Excellence Grant, NIH/NCRR/RCMI G12-RR03034, and the MBRS/RISE Program 2R25GM058268.
Citation Format: Kisha A. Scarlett, Vaishnavi Pattabiraman, Leonard M. Anderson. Iroquois homeobox gene 3 is temporally regulated in response to vascular endothelial growth factor and modulates endothelial cell migration and tubulogenesis. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C38.