title: Multiplexed digital quantification of binge-like alcohol-mediated alterations in maternal uterine angiogenic mRNA transcriptome.
abstract: Genomic studies on fetal alcohol spectrum disorders (FASD) have utilized either genome-wide microarrays/bioinformatics or targeted real-time PCR (RT-PCR). We utilized herein for the first time a novel digital approach with high throughput as well as the capability to focus on one physiological system. The aim of the present study was to investigate alcohol-induced alterations in uterine angiogenesis-related mRNA abundance using digital mRNA technology. Four biological and three technical replicates of uterine arterial endothelial cells from third-trimester ewes were fluorescence-activated cell sorted, validated, and treated without or with binge-like alcohol. A capture probe covalently bound to an oligonucleotide containing biotin and a color-coded reporter probe were designed for 85 angiogenesis-related genes and analyzed with the Nanostring nCounter system. Twenty genes were downregulated (↓) and two upregulated (↑), including angiogenic growth factors/receptors (↓placental growth factor), adhesion molecules (↓angiopoietin-like-3; ↓collagen-18A1; ↓endoglin), proteases/matrix proteins/inhibitors (↓alanyl aminopeptidase; ↓collagen-4A3; ↓heparanase; ↓plasminogen, ↑plasminogen activator urokinase; ↓platelet factor-4; ↓plexin domain containing-1; ↓tissue inhibitor of metalloproteinases-3), transcription/signaling molecules (↓heart and neural crest derivatives-2; ↓DNA-binding protein inhibitor; ↓NOTCH-4; ↓ribosomal protein-L13a1; ↓ribosomal protein large-P1), cytokines/chemokines (↓interleukin-1B), and miscellaneous growth factors (↓leptin; ↓platelet-derived growth factor-α); ↓transforming growth factor (TGF-α; ↑TGF-β receptor-1). These novel data show significant detrimental alcohol effects on genes controlling angiogenesis supporting a mechanistic role for abnormal uteroplacental vascular development in FASD. The tripartite digital gene expression system is therefore a valuable tool to answer many additional questions about FASD from both mechanistic as well as ameliorative perspectives.
authors.affiliation: Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas 77555, USA. jaramado@utmb.edu
authors.forename: Jayanth
authors.initials: J
authors.lastname: Ramadoss
authors.forename: Ronald R
authors.initials: RR
authors.lastname: Magness
mesh_terms: D000437:Alcoholism / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D000431:Ethanol / Q000494:pharmacology; D005260:Female; D009389:Neovascularization, Pathologic / Q000235:genetics* / Q000378:metabolism; D011247:Pregnancy; D011248:Pregnancy Complications / Q000235:genetics* / Q000378:metabolism; D023121:RNA, Messenger, Stored / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D012756:Sheep; D059467:Transcriptome; D014599:Uterus / Q000098:blood supply / Q000187:drug effects*
chemical_list: D023121:RNA, Messenger, Stored; D000431:Ethanol