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.

摘要

BACKGROUND: Pregnancy-induced utero-placental growth, angiogenic remodeling, and enhanced vasodilation are all partly regulated by estradiol-17beta-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 (FASDs). In this study, we hypothesized that in vitro chronic binge-like alcohol will decrease uterine arterial endothelial eNOS expression and alter its multisite 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-17beta-induced uterine angiogenesis in vitro. METHODS: Uterine artery endothelial cells were isolated from pregnant ewes (gestational day 120 to 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 hours 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 2 days, and then treated with increasing concentrations of estradiol-17beta (0.1, 1, 10, 100 nM) in the absence or presence of LD or HD alcohol to evaluate estradiol-17beta-induced angiogenesis index using BrdU proliferation assay. RESULTS: LD and HD binge-like alcohol decreased uterine arterial eNOS expression (p = 0.009). eNOS multisite phosphorylation activation state was altered: P(635) eNOS was decreased (p = 0.017), P(1177) eNOS was not altered, and P(495) 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 posttranslational modifications. Assessment of uterine vascular adaptation via angiogenesis demonstrated that alcohol abrogated the dose-dependent proliferative effects of estradiol-17beta and thus blunted angiogenesis. CONCLUSIONS: Thus, the maternal uterine vasculature during pregnancy may be vulnerable to chronic binge-like alcohol. Altered eNOS multisite phosphorylation also suggests that alcohol produces specific effects at the level of posttranslational modifications critical for pregnancy-induced uterine vascular adaptations. Finally, the alcohol and estradiol-17beta data suggest a negative impact of alcohol on estrogen actions on the uterine vasculature.