title: The NADH oxidase ENOX1, a critical mediator of endothelial cell radiosensitization, is crucial for vascular development.
abstract: 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 hybridization coupled with immunofluorescence performed on zebrafish embryos demonstrate that enox1 message and 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 that morpholino-mediated knockdown of enox1 increases NADH levels, consistent with loss of enzyme. VJ115 is a small-molecule inhibitor of Enox1's oxidase activity shown to increase intracellular NADH in endothelial cells; 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.
authors.affiliation: Authors' Affiliations: Departments of Radiation Oncology and Medicine and Cell & Developmental Biology; Genomic Sciences Resources, Vanderbilt University Medical Center; Biomedical Engineering, Vanderbilt School of Engineering, Nashville, Tennessee; School of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, Arkansas; and Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California.
authors.forename: Amudhan
authors.initials: A
authors.lastname: Venkateswaran
authors.forename: Konjeti R
authors.initials: KR
authors.lastname: Sekhar
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Levic
authors.forename: David B
authors.initials: DB
authors.lastname: Melville
authors.forename: Travis A
authors.initials: TA
authors.lastname: Clark
authors.forename: Witold M
authors.initials: WM
authors.lastname: Rybski
authors.forename: Alexandra J
authors.initials: AJ
authors.lastname: Walsh
authors.forename: Melissa C
authors.initials: MC
authors.lastname: Skala
authors.forename: Peter A
authors.initials: PA
authors.lastname: Crooks
authors.forename: Ela W
authors.initials: EW
authors.lastname: Knapik
authors.forename: Michael L
authors.initials: ML
authors.lastname: Freeman
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D004730:Endothelium, Vascular / Q000196:embryology* / Q000201:enzymology / Q000254:growth & development*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D018919:Neovascularization, Physiologic / Q000502:physiology; D015027:Zebrafish
chemical_list: D009097:Multienzyme Complexes; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases