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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.

SUBMITTER: Ramadoss J

PROVIDER: S-EPMC3426435 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Multiplexed digital quantification of binge-like alcohol-mediated alterations in maternal uterine angiogenic mRNA transcriptome.

Ramadoss Jayanth J Magness Ronald R RR

Physiological genomics 20120424 11

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 replica ...[more]

PMID: 22535877

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Project description:We hypothesized that maternal nutrient restriction starting at the time of breeding would influence placental vascular development and gene expression of angiogenic factors during the first 50 d of gestation in beef heifers. Commercial Angus crossbred heifers (n = 49) were maintained on a total mixed ration and supplemented with dried distillers grains with solubles. All heifers were subject to 5-d CO-Synch + CIDR estrous synchronization protocol, AI to a single Angus sire, and randomly assigned to dietary treatments. One half were assigned to control diet (CON) targeted to gain 0.45 kg/d and the remaining half were assigned to restricted diet (RES), which received 60% of CON. Heifers were subjected to ovariohysterectomy on d 16, 34, or 50 of gestation. Utero-placental tissues were obtained from the uterine horns ipsilateral and contralateral to the corpus luteum and separated into maternal caruncle (CAR); maternal endometrium, inter-caruncle (ICAR), and fetal membranes (FM). After collection, all tissues were snap frozen and stored at -80°C. There were no treatment × stage of gestation interactions (P >0.13) on the mRNA expression of vascular endothelial growth factor (VEGF) or endothelial nitric oxide synthase (eNOS). Heifers on CON treatment had greater (P = 0.03) expression of VEGF compared with RES heifers in NP-ICAR. On d 50 expression of eNOS was increased (P = 0.05) compared with d 16 in P-CAR. Expression of eNOS mRNA was decreased (P = 0.04) on d 16 compared with d 34 and 50 in CON heifer. Gene expression of eNOS was increased (P < 0.001) in the pregnant uterine horn compared with the NP uterine horn on d 34 and 50. Expression of eNOS was also increased (P < 0.003) on d 34 and 50 in the pregnant uterine horn compared with FM. There was a maternal nutritional plane × stage of gestation interaction (P = 0.01) on the vascular ratio (vascular volume/tissue volume) in maternal tissues. The RES heifers had a greater vascular ratio on d 16 compared with d 34 and 50; whereas, CON heifers had a greater vascular ratio on d 34 compared with d 16 and 50. In the NP uterine horn, there was also an increase (P = 0.02) in vascular volume of FM from CON heifers compared with FM from RES heifers. We conclude that maternal nutrient restriction did alter both vascularity and mRNA expression of angiogenic factor in utero-placental tissues during the establishment of pregnancy in first parity beef heifers.

Project description:BackgroundPrenatal alcohol exposure (PAE) is associated with postnatal iron deficiency (ID), which has been shown to exacerbate deficits in growth, cognition, and behavior seen in fetal alcohol spectrum disorders. However, the mechanisms underlying PAE-related ID remain unknown.ObjectivesWe aimed to examine biochemical measures of iron homeostasis in the mother, placenta, neonate, and 6.5-month-old infant.MethodsIn a prenatally recruited, prospective longitudinal birth cohort in South Africa, 206 gravidas (126 heavy drinkers and 80 controls) were interviewed regarding alcohol, cigarette, and drug use and diet at 3 prenatal visits. Hemoglobin, ferritin, and soluble transferrin receptor (sTfR) were assayed twice during pregnancy and urinary hepcidin:creatinine was assayed once. Infant ferritin and hemoglobin were measured at 2 weeks and 6.5 months and sTfR was measured at 6.5 months. Histopathological examinations were conducted on 125 placentas and iron transport assays (iron regulatory protein-2, transferrin receptor-1, divalent metal transporter-1, ferroportin-1, and iron concentrations) were conducted on 63.ResultsIn multivariable regression models, prenatal drinking frequency (days/week) was related to higher maternal hepcidin and to sequestration of iron into storage at the expense of erythropoiesis in mothers and neonates, as evidenced by a lower hemoglobin (g/dL)-to-log(ferritin) (ug/L) ratio [mothers: raw regression coefficient (β) = -0.21 (95% CI: -0.35 to -0.07); neonates: β = -0.15 (95% CI: -0.24 to -0.06)]. Drinking frequency was also related to decreased placental ferroportin-1:transferrin receptor-1 (β = -0.57 for logged values; 95% CI: -1.03 to -0.10), indicating iron-restricted placental iron transport. At 6.5 months, drinking frequency was associated with lower hemoglobin (β = -0.18; 95% CI: -0.33 to -0.02), and increased prevalences of ID (β = 0.09; 95% CI: 0.02-0.17) and ID anemia (IDA) (β = 0.13; 95% CI: 0.04-0.23). In causal inference analyses, the PAE-related increase in IDA was partially mediated by decreased neonatal hemoglobin:log(ferritin), and the decrease in neonatal hemoglobin:log(ferritin) was partially mediated by decreased maternal hemoglobin:log(ferritin).ConclusionsIn this study, greater PAE was associated with an unfavorable profile of maternal-fetal iron homeostasis, which may play mechanistic roles in PAE-related ID later in infancy.

Project description:Background and purposeThe ability to track changes in gene expression following viral infection is paramount to understanding viral pathogenesis. This study was undertaken to evaluate the nCounter, a high throughput digital gene expression system, as a means to better understand West Nile virus (WNV) dissemination and the inflammatory response against WNV in the outbred Swiss Webster (SW) mouse model over the course of infection.MethodologyThe nCounter Mouse Inflammation gene expression kit containing 179 inflammation related genes was used to analyze gene expression changes in multiple tissues over a nine day course of infection in SW mice following intraperitoneal injection with WNV. Protein expression levels for a subset of these cytokine/chemokine genes were determined using a multiplex protein detection system (BioPlex) and comparisons of protein/RNA expression levels made.ResultsExpression analysis of spleen, lung, liver, kidney and brain of SW mice infected with WNV revealed that Cxcl10 and Il12b are differentially expressed in all tissues tested except kidney. Data stratification of positively confirmed infected (WNV (+)) versus non-infected (WNV (-) tissues allowed differentiation of the systemic inflammatory gene response from tissue-specific responses arising from WNV infection. Significant (p<0.05) decrease in C3ar1 was found in WNV (-) spleen. Il23a was significantly upregulated, while Il10rb was down-regulated in WNV (-) lung. Il3 and Mbl2 were down-regulated in WNV (-) liver. In WNV (+) livers, Stat1, Tlr2, chemokines Cxcl1, Cxcl3, Cxcl9, Cxcl10, cytokines Il6, Il18, cytokine-related gene Il1r and cytokine agonist Ilrn were significantly upregulated. In WNV (-) brain tissues, Csf2 and Cxcl10 were significantly upregulated. Similar gene and protein expression kinetics were found for Ccl2, Ccl3, Ccl4 and Ccl5 and correlated with the presence of infectious virus. In summary, the utility of the nCounter platform for rapid identification of gene expression changes in SW mice associated with WNV infection was demonstrated.

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Multiplexed digital quantification of binge-like alcohol-mediated alterations in maternal uterine angiogenic mRNA transcriptome.

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Multiplexed digital quantification of binge-like alcohol-mediated alterations in maternal uterine angiogenic mRNA transcriptome.

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