The NADH Oxidase ENOX1, a Critical Mediator of Endothelial Cell Radiosensitization, Is Crucial for Vascular Development

ENOX1 is a highly conserved NADH oxidase that helps to regulate intracellular nicotinamide adenine dinucleotide levels in many cell types, including endothelial cells. Pharmacologic and RNA interference (RNAi)–mediated suppression of ENOX1 impairs surrogate markers of tumor angiogenesis/vasculogenesis, providing support for the concept that ENOX1 represents an antiangiogenic druggable target. However, direct genetic evidence that demonstrates a role for ENOX1 in vascular development is lacking. In this study, we exploited a zebrafish embryonic model of development to address this question. Whole-mount in situ hybridizationcoupledwithimmunofluorescenceperformedonzebrafishembryosdemonstratethatenox1messageand translated protein are expressed in most tissues, and its expression is enriched in blood vessels and heart. Morpholino-mediated suppression of Enox1 in Tg(fli1-eGFP) and Tg(flk1-eGFP) zebrafish embryos significantly impairs the development of vasculature and blood circulation. Using in vivo multiphoton microscopy, we show thatmorpholino-mediatedknockdownofenox1increasesNADHlevels,consistentwithlossofenzyme.VJ115isa small-moleculeinhibitorofEnox1 0 soxidaseactivityshowntoincreaseintracellularNADHinendothelialcells;we used VJ115 to determine if the oxidase activity was crucial for vascular development. We found that VJ115 suppressed vasculogenesis in Tg(fli1-eGFP) embryos and impaired circulation. Previously, it was shown that suppression of ENOX1 radiosensitizes proliferating tumor vasculature, a consequence of enhanced endothelial cell apoptosis. Thus, our current findings, coupled with previous research, support the hypothesis that ENOX1 represents a potential cancer therapy target, one that combines molecular targeting with cytotoxic sensitization. Cancer Res; 74(1); 38–43. � 2013 AACR.