Alcohol and Maternal Uterine Vascular Adaptations During Pregnancy-Part I: Effects of Chronic In Vitro Binge-Like Alcohol on Uterine Endothelial Nitric Oxide System and Function

Abstract

Background Pregnancy-induced utero-placental growth, angiogenic remodeling, and enhanced vasodilation are all partly regulated by estradiol-17β-mediated activation of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production. However, very little is known about the effects of alcohol on these maternal utero-placental vascular adaptations during pregnancy and its potential role in the pathogenesis of Fetal Alcohol Spectrum Disorders (FASD). In this study, we hypothesized that in vitro chronic binge-like alcohol will decrease uterine arterial endothelial eNOS expression and alter its multi-site phosphorylation activity state via disruption of AKT signaling. To study the direct effects of alcohol on uterine vascular adaptations, we further investigated the effects of alcohol on estradiol-17β-induced uterine angiogenesis in vitro. Methods Uterine artery endothelial cells were isolated from pregnant ewes (gestational day 120-130; term = 147), Fluorescence Activated Cell sorted, validated, and maintained in culture to passage 4. To mimic maternal binge drinking patterns, cells were cultured in the absence or presence of a lower (LD) or higher dose (HD) of alcohol in a compensating sealed humidified chamber system equilibrated with aqueous alcohol for 3 h on 3 consecutive days. Immunoblotting was performed to assess expression of NO system-associated proteins and eNOS multi-site phosphorylation. Following this treatment paradigm, control and binge alcohol treated cells were passaged, grown for two days, and then treated with increasing concentrations of estradiol-17β (0.1, 1, 10, 100 nM) in the absence or presence of LD or HD alcohol to evaluate estradiol-17β-induced angiogenesis index using BrdU Proliferation Assay. Results LD and HD binge-like alcohol decreased uterine arterial eNOS expression (P=0.009). eNOS multi-site phosphorylation activation state was altered: P635eNOS was decreased (P=0.017), P1177 eNOS was not altered, and P495 eNOS exhibited an inverse U shaped dose-dependent relationship with alcohol. LD and HD alcohol decreased the major eNOS-associated protein cav-1 (P<0.001). However, the commonly implicated AKT pathway did not correlate with eNOS post-translational modifications. Assessment of uterine vascular adaptation via angiogenesis demonstrated that alcohol abrogated the dose-dependent proliferative effects of estradiol-17β and thus blunted angiogenesis. Conclusions Thus, the maternal uterine vasculature during pregnancy may be vulnerable to chronic binge-like alcohol. Altered eNOS multi-site phosphorylation also suggests that alcohol produces specific effects at the level of post-translational modifications critical for pregnancy-induced uterine vascular adaptations. Finally, the alcohol and estradiol-17β data suggest a negative impact of alcohol on estrogen actions on the uterine vasculature.

Effects of l-glutamine supplementation on maternal and fetal hemodynamics in gestating ewes exposed to alcohol

“…A study in pregnant C57BL/6 J mice demonstrated that NO modulation of the systemic mesenteric artery vascular response was hampered due to alcohol exposure (Cook et al 2001). In vitro studies have shown that binge-like alcohol exposure impairs eNOS activation and reduces mRNA levels for angiogenic genes and related proteome in endothelial cells of the ovine uterine artery, hence blunting the uterine vascular adaptations, including vasodilatory and angiogenic pathways (Ramadoss et al 2010, 2011; Ramadoss and Magness 2011, 2012a, b, d). Finally, these findings have significant implications on fetal growth and development.…”

“…Unfortunately, the impact of maternal alcohol exposure on uterine perfusion has received minimal attention and there is a paucity of information in the literature. An earlier study in sheep has shown that acute alcohol exposure leads to a significant reduction in uterine blood flow (Falconer 1990) and a series of in vitro studies have shown that binge-like alcohol exposure alters utero-placental vascular adaptations (Ramadoss et al 2011, 2012; Ramadoss and Magness 2011, 2012a, b; Subramanian et al 2014). However, to our knowledge, so far no study has been conducted to investigate the effect of repeated binge alcohol exposure during pregnancy on uterine blood flow.…”

Vascular effects of maternal alcohol consumption

“…In a chick extraembryonic model, moderate and heavy alcohol doses (30% or 50%, 24 or 48 h) impaired vascular development, and the authors (89) have attributed these deficits to angiogenic growth factors, associated receptors, and oxidative stress, whereas Gu et al (29), again using the chick extraembryonic model, showed that administration of alcohol (0.25 g·kg Ϫ1 ·day Ϫ1 , 7 days) induced greater branching of blood vessels (29). In uterine artery endothelial cells derived from pregnant ewes, in vitro chronic binge-like alcohol exposure (300 or 600 mg/dl) inhibits estradiol 17␤-induced endothelial proliferation (67). In a more recent study, biotinylated capture and color-coded reporter probes were used to digitally characterize 85 angiogenesisassociated genes in the uterine vasculature following chronic binge alcohol exposure (22,26).…”

“…In one study, alcohol administered in the diet (GD 6 -18, peak BAC, 0.11 g/dl) to pregnant C57BL/6J mice showed reduced NO modulation of the mesenteric artery vascular response (14). In vitro chronic binge-like alcohol treatment (300 and 600 mg/dl) to uterine artery endothelial cells derived from third trimester pregnant ewes decreased excitatory serine-635 endothelial NO synthase phosphorylation levels at both concentrations (67). In placental villi, in vitro acute alcohol exposure for 2 h (100 and 200 mM) decreased tissue NO concentrations and cGMP levels and increased superoxide dismutase activity (39).…”

Chronic binge alcohol consumption during pregnancy alters rat maternal uterine artery pressure response

“…We and others have reported that alcohol consumption during pregnancy results in altered uterine endothelial adaptations (Ramadoss, Jobe, & Magness, 2011; Ramadoss & Magness, 2011, 2012a, 2012c), agonist-dependent vessel relaxation (Subramanian et al, 2014), spiral artery remodeling (Gundogan et al, 2008), and blood flow (Falconer, 1990). However, there exists no study on alcohol-related myogenic response, a critical gestational uterine vascular adaptation.…”