Abstract 384: NADH oxidase, Enox1 activity, is required for vasculogenesis in Zebrafish.

Abstract

Abstract Establishment of an adequate vascular network is a crucial event for tumor development. Emerging research has implicated the endothelial redox state as a significant regulator of vascular growth and remodeling. ENOX1 encodes a protein that is expressed in mammalian endothelial cells and exhibits NADH oxidase activity. Recent research suggests that ENOX1 functions as a positive regulator of angiogenesis. However, direct genetic evidence supporting this hypothesis is lacking. We used a zebrafish model to directly examine the role of Enox1 during embryonic vascular development. Whole mount in situ hybridization coupled with immunocytochemistry performed on zebrafish cryosections show that enox1 message and the translated protein are expressed in heart and blood vessels during embryonic development. Furthermore, use of immunocytochemistry demonstrates that Enox1 co-localizes with vascular expression of the ETS-domain transcription factor fli in heart and intersegmental vessels. Using in vivo multiphoton microscopy, we show that morpholino-mediated knockdown of Enox1 in Zebrafish embryos increases NADH fluorescence, an expected consequence of loss of Enox1 oxidase activity. Phenotypic manifestations resulting from loss of Enox1 function in embryos include vascular defects, most notably loss of fli-eGFP and flk-eGFP (receptor kdr, VEGFR2 in mammals) expression in intersegmental vessels and a loss of blood circulation in the intersegmental vessels. Pharmacological inhibition of Enox1 activity using a novel small molecule phenocopies the defects observed following morpholino-mediated functional knockdown. Together, these results show that Enox1-mediated redox regulation is essential for vasculogenesis in vertebrates in vivo. These results, in conjunction with previous work that demonstrated that inhibition of ENOX1 radiosensitized tumor vasculature, support the hypothesis that ENOX1 represents a novel target for impairing tumor angiogenesis. Citation Format: Amudhan Venkateswaran, Daniel S. Levic, David B. Melville, Konjeti R. Sekhar, Alexandra J. Walsh, Melissa C. Skala, Peter A. Crooks, Ela W. Knapik, Michael L. Freeman. NADH oxidase, Enox1 activity, is required for vasculogenesis in Zebrafish. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 384. doi:10.1158/1538-7445.AM2013-384