Arsenic Exposure Inhibits Angiogenesis in Zebrafish via Downregulation of both VEGFA and VEGFR2 Sherry G. Clendeno 1 , Divya Ganapathi Sankaran 1 , Abbas Shirinifard 1,2 , Catherine W. McCollum 3 , Maria Bondesson Bolin 3 , Jan-Åke Gustafsson 3 , James A. Glazier 1 1. Biocomplexity Institute, Department of Physics, Indiana University, Bloomington, Indiana 2. Information Sciences, St. Jude Children’s Research Hospital, Memphis, Tennessee 3. Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, Houston, Texas Zebrafish intersegmental vessel (ISV) sprouting is a common animal model for study of angiogenesis and for screening of toxins and drugs that affect angiogenesis [1,2]. Zebrafish embryos develop rapidly, with onset of gastrulation at 6 hours post fertilization (hpf), somitogenesis at 10.5 hpf and vascular development at 14 hpf [3]. Angioblasts migrate to the midline to form the dorsal aorta (DA) and, subsequently, the posterior cardinal vein by vasculogenesis. The notochord initiates angiogenic sprouting from these primary vessels by secreting sonic hedgehog (SHH), which upregulates vascular endothelial growth factor A (VEGFA) expression and secretion in adjacent myotomes. Secreted VEGFA is present in free (VEGFA121) and extracellular-matrix (ECM)-bound forms (VEGFA165). In zebrafish, only VEGFA165, promotes sprouting [4]. Endothelial cells (ECs) of the DA, express VEGF receptor 2 (VEGFR2), bind VEGFA and migrate up VEGFA gradients in the ECM [5]. Migrating ECs in an angiogenic sprout assume either tip or stalk phenotypes [5]. In tip ECs, binding of VEGFA to VEGFR2 upregulates VEGFR2 and Dll4 expression and downregulates Notch expression [6]. Tip-cell Dll4 expression upregulates Notch expression in adjacent ECs, which then downregulates VEGFR2 and Dll4 expression, inducing the stalk phenotype. Additional factors further modulate this core regulatory mechanism [1]. Arsenic (As), an environmental toxin, inhibits angiogenic ISV sprouting in zebrafish, resulting in shorter length, irregularly oriented, or entirely missing ISVs [2,7]. Our analyses of ISV growth dynamics showed that these changes result from decreased directed migration speed and perturbation of directional path-finding [7]. In cell culture, As exposure elevates reactive oxygen species and VEGFA, disrupts cell-cell junctions and affects Notch signaling, a key regulator of VEGFR2 [8]. To determine how As inhibits angiogenic sprouting in vivo, we quantified levels of the chemo-attractant VEGFA and its receptor VEGFR2 in normal ISV sprouting and under As exposure. We then developed a mechanistic computer simulation of angiogenic ISV sprouting to assess the sufficiency of these hypothesized modes of action of As. We immuno-labeled VEGFA165 and VEGFR2 in whole zebrafish embryos and evaluated protein expression levels using three-dimensional (3D) quantitative confocal microscopy. We counterstained surrounding tissue with lens-culinaris-agglutinin (LCA). We fixed control and As-exposed (100 μg/ml and 400 μg/ml) flt1-eGFP (flt1 is VEGFR2) embryos overnight in 4% paraformaldehyde in PBS at 18, 19, 20, 22, 24 hpf, then blocked and permeablized overnight in 1% BSA, 5% horse serum, 1% Triton-X-100 in PBS. We then incubated embryos overnight in anti-zebrafish VEGFA165 (R&D Systems, Minneapolis, MN, USA) at 1:100, FITC anti-GFP (Invitrogen, Grand Island, NY, USA) at 1:100, and rhodamine conjugated LCA (Vector Labs, Burlingame, CA, USA) at 1:50, followed by overnight incubation in AF633-anti-mouse secondary antibody (Invitrogen) at 1:50, all diluted in 5% BSA, 1% HS, in PBS. We acquired 3D 778 doi:10.1017/S1431927613005886 Microsc. Microanal. 19 (Suppl 2), 2013 © Microscopy Society of America 2013 https://doi.org/10.1017/S1431927613005886 Downloaded from https://www.cambridge.org/core. IP address: 54.162.69.248, on 16 Jun 2020 at 08:14:05, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms.