title: Exposure of chick embryos to cadmium changes the extra-embryonic vascular branching pattern and alters expression of VEGF-A and VEGF-R2.
abstract: Cadmium (Cd) has several industrial applications, and is found in tobacco products, a notable source of human exposure. Vascular endothelial cells are key targets of Cd toxicity. Here, we aim to quantify the alteration to vascular branching pattern following Cd exposure in the chick extra-embryonic membrane (EEM) using fractal analysis, and explore molecular cues to angiogenesis such as VEGF-A and VEGF-R2 expression following Cd treatment. Chicken embryos were incubated for 60 h to Hamburger-Hamilton developmental stage 16-17, then explanted and treated with 50 μL of 50 μmol cadmium acetate (CdAc) or an equivalent volume of equimolar sodium acetate (NaAc). Images of embryos and their area vasculosa (AV) were captured and analyzed at 4 different time points (4, 8, 24 and 48 h) following treatment. Vascular branching in the AV was quantified using its fractal dimension (Df), estimated using a box counting method. Gallinaceous VEGF ELISA was used to measure the VEGF-A concentration in the EEM following treatment, with determination of the relative expression of VEGF-A and VEGF-R2 using quantitative real-time RT-PCR. Vascular branching increased monotonically in the control group at all time points. The anti-angiogenic effect of Cd exposure on the AV was reflected by a significant reduction in Df when compared with controls. Df was more markedly reduced in cultures with abnormal embryos. The expression of VEGF-A protein, and VEGF-A and VEGF-R2 mRNA were reduced in Cd-exposed EEMs. Both molecules contribute to growth, vessel sprouting and branching processes, which supports our findings using fractal analysis.
authors.affiliation: School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Anna Kaskova
authors.initials: AK
authors.lastname: Gheorghescu
authors.affiliation: School of Physics, University College Dublin, Belfield, Dublin 4, Ireland; Complex and Adaptive Systems Laboratory, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Bartlomiej
authors.initials: B
authors.lastname: Tywoniuk
authors.affiliation: School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4, Ireland; National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
authors.forename: Johannes
authors.initials: J
authors.lastname: Duess
authors.affiliation: School of Physics, University College Dublin, Belfield, Dublin 4, Ireland; Complex and Adaptive Systems Laboratory, University College Dublin, Belfield, Dublin 4, Ireland. Electronic address: buchete@ucd.ie.
authors.forename: Nicolae-Viorel
authors.initials: NV
authors.lastname: Buchete
authors.affiliation: School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4, Ireland. Electronic address: jennifer.thompson@ucd.ie.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Thompson
mesh_terms: D000085:Acetates / Q000633:toxicity*; D020533:Angiogenesis Inhibitors / Q000633:toxicity; D000818:Animals; D002104:Cadmium / Q000633:toxicity*; D002642:Chick Embryo / Q000187:drug effects* / Q000196:embryology* / Q000378:metabolism; D002645:Chickens; D005321:Extraembryonic Membranes / Q000187:drug effects / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D007091:Image Processing, Computer-Assisted; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism*
chemical_list: D000085:Acetates; D020533:Angiogenesis Inhibitors; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D002104:Cadmium; C028031:cadmium acetate; D040301:Vascular Endothelial Growth Factor Receptor-2
keywords: Angiogenesis; Area vasculosa; Cadmium; Chick embryo; Fractal analysis