Background: Transcription regulation is essential for angiogenesis, but the role of Irx3 in this process remains to be defined.Results: Irx3 promotes endothelial cell migration and tip cell specification through VEGF-Notch signaling.Conclusion: Irx3 is a novel proangiogenic mediator of endothelial cell migration and cell fate.Significance: Manipulation of Irx3 may provide novel therapeutic strategies in adult vascular pathologies.

The Iroquois homeobox (Irx5) gene is essential in embryonic development and cardiac electrophysiology. Although recent studies have reported that IRX5 protein is involved in regulation of the cell cycle and apoptosis in prostate cancer cells, little is known about the role of IRX5 in the adult vasculature. Here we report novel observations on the role of IRX5 in adult vascular smooth muscle cells (VSMCs) during proliferation in vitro and in vivo. Comparative studies using primary human endothelial cells, VSMCs, and intact carotid arteries to determine relative expression of Irx5 in the peripheral vasculature demonstrate significantly higher expression in VSMCs. Sprague-Dawley rat carotid arteries were subjected to balloon catherization, and the presence of IRX5 was examined by immunohistochemistry after 2 wk. Results indicate markedly elevated IRX5 signal at 14 days compared with uninjured controls. Total RNA was isolated from injured and uninjured arteries, and Irx5 expression was measured by RT-PCR. Results demonstrate a significant increase in Irx5 expression at 3–14 days postinjury compared with controls. Irx5 genetic gain- and loss-of-function studies using thymidine and 5-bromo-2′-deoxyuridine incorporation assays resulted in modulation of DNA synthesis in primary rat aortic VSMCs. Quantitative RT-PCR results revealed modulation of cyclin-dependent kinase inhibitor 1B (p27kip1), E2F transcription factor 1 (E2f1), and proliferating cell nuclear antigen (Pcna) expression in Irx5-transduced VSMCs compared with controls. Subsequently, apoptosis was observed and confirmed by morphological observation, caspase-3 cleavage, and enzymatic activation compared with control conditions. Taken together, these results indicate that Irx5 plays an important role in VSMC G1/S-phase cell cycle checkpoint control and apoptosis.

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.

Purpose: 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, and decreasing the health disparity gap in terms of cancer treatment. 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. Citation Format: Kisha Scarlett, Pattabiraman Vaishnavi, Leonard Anderson. Iroquois homeobox gene 3 is temporally regulated in response to vascular endothelial growth factor and modulates endothelial cell migration. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C64. doi:10.1158/1538-7755.DISP13-C64