2-D DIGE uterine endothelial proteomic profile for maternal chronic binge-like alcohol exposure

Abstract

Little is known about alcohol effects on the utero-placental compartment during pregnancy. For the first time, we utilized 2-D DIGE quantitative proteomics to evaluate the role of the uterus in Fetal Alcohol Spectrum Disorders (FASD) pathogenesis. Uterine artery endothelial cells were isolated from pregnant ewes, FAC sorted, validated, and maintained in culture. To mimic maternal binge drinking patterns, cells were cultured in the absence or presence of alcohol (300 mg/dl) in a compensating sealed humidified chamber system equilibrated with aqueous alcohol for 3 h on 3 consecutive days for two weeks. CyDye switch combined with 2-D DIGE followed by MALDI-TOF and tandem MS/MS were utilized. Validation was performed using Western immunoblot analysis. Chronic binge-like alcohol significantly (P < 0.05) decreased 30 proteins and increased 19 others. Gene-enrichment and functional annotation cluster analysis revealed significant enrichment (P < 0.05) in three categories: glutathione S transferase, thioredoxin, and vesicle transport-related. Furthermore, alcohol differentially altered proteins with certain isoforms being downregulated while others were upregulated. In summary, binge alcohol has specific effects on the maternal uterine proteome, especially those related to oxidative stress. The current study also demonstrates a great need to utilize proteomic approaches for diagnostic, mechanistic and therapeutic aspects of FASD.

Fetal alcohol spectrum disorders and cognitive functions of young children

“…Antenatal ethanol exposure induces oxidative stress in various organs (Amini et al, 1996;Ramadoss and Magness, 2011;Patten et al, 2013). The fetal brain is affected by this type of stress even when the consumption of alcohol is relatively low (Chu et al, 2007;Dong et al, 2010).…”

Vascular effects of maternal alcohol consumption

“…Next, we will discuss oxygen free radicals as redox balance impairment produces vasoconstriction. Substantial evidence exists to demonstrate maternal alcohol exposure increases oxidative stress markers in the fetus (11), fetoplacental compartment (40), maternal ovine uterine artery endothelial cells (69), etc., via multiple mechanisms that are discussed in other excellent review articles (9,11,27), whereas there is a paucity of human clinical studies especially in pregnant women (79). Another paracrine factor that is critical in gestational adaptations is NO.…”

“…This is an important area of exploration since the vascular endothelium is programmed during pregnancy (47,48); for example, we reported that 17␤-estradiol and its metabolites induce proliferation of uterine endothelial cells only during pregnancy and not in the nonpregnant state (37). In one study, uterine artery endothelial cells were isolated from pregnant ewes, fluorescence-activated cell sorted, validated, and maintained in culture and treated with chronic in vitro binge-like alcohol (300 mg/dl, 3-h treatment on 3 consecutive days followed by 4 days without exposure for 2 wk) (69). Two-dimensional differential in gel electrophoresis separation followed matrix-assisted laser desorption time-of-flight mass spectrometry tandem mass spectrometry was then used to demonstrate that alcohol significantly decreased 30 proteins and increased 19 others in the uterine endothelium (Fig.…”

“…3). Gene enrichment and functional annotation cluster analysis was then used to show significant enrichment in three categories: oxidative stress-associated glutathione S-transferase and thioredoxin, as well as vesicle transport-related pathways (69). It has also been demonstrated that alcohol significantly alters the protein expression in the uterine artery endothelial caveolar lipid rafts where numerous receptors, signaling molecules, ion channels, etc., reside (28,60,68).…”

“…C: green spots on the 2-D gel depict maternal uterine endothelial proteins that were downregulated by alcohol, whereas red spots were proteins that were upregulated and yellow spots were unaltered by binge alcohol exposure. Adapted with permission from Ramadoss and Magness (69). dynamic variables such as blood pressure and total peripheral resistance and how these variables are altered by baro-and chemoreflexes.…”