Prenatal alcohol exposure disrupts miR‐150‐5p regulation of angiogenesis and blood‐brain barrier integrity genes

Abstract

Fetal alcohol spectrum disorders (FASD) occur after in utero ethanol (EtOH) exposure and manifest in a wide range of neurocognitive deficits. These deficits can be caused by EtOH-induced dysregulation of genes required for angiogenesis and vascular integrity which are being regulated by microRNAs (miRNAs). miRNAs silence gene expression by binding to the 3’UTRs of target mRNAs, inducing mRNA decay. In an established mouse model of moderate prenatal alcohol exposure (PAE), we found that cortices (CTX) and primary brain microvascular endothelial cells (pBMVECs) isolated from PAE pups at embryonic day 18 (E18) had significantly higher levels of miR-150-5p compared to saccharin-exposed controls. A novel angiogenic target of miR-150-5p, vascular endothelial zinc finger 1 (Vezf1), is an endothelial-specific transcription factor required for vascular development. Vezf1 levels were significantly reduced in E18 PAE CTX and pBMVECs compared to controls. pBMVECs also showed a decrease in expression of other genes important for vascular function and integrity, including Claudin-12, Claudin-5, Occludin, VE-cadherin and MMP14. To identify the effects of EtOH on endothelial cell gene expression, we treated a mouse BMVE cell line (BMVECs) with 25mM of EtOH for 24 hrs. miR-150-5p expression increased after EtOH treatment compared to no treatment controls. Transfection of miR-150-5p LNATM mimics in BMVECs decreased Vezf1, Claudin-12 and MMP14 expression, while miR-150-5p LNATM inhibitors increased their expression. To demonstrate direct interaction between miR-150-5p and Vezf1, we cloned the Vezf1 3’UTR into a firefly luciferase reporter. Dual luciferase assays showed reduced luciferase activity for the Vezf1 3’UTR reporter with miR-150-5p LNATM mimics. To investigate the effect of EtOH and miR-150-5p on endothelial cell function, we performed in vitro migration, tube formation and permeability assays. BMVECs treated with EtOH significantly attenuated migration and tube formation vs no EtOH treatment controls. In addition, miR-150-5p overexpression in BMVECs attenuated tube formation and cell migration, and significantly increased cell permeability. Correspondingly, miR-150-5p inhibition significantly enhanced tube formation and migration, and decreased cell permeability. Our research suggests that miR-150-5p may adversely alter the cortical microvasculature during PAE by inhibiting the expression of targets important for angiogenesis and blood-brain barrier integrity.