Ethanol Alters DNMT1/3a/3b Expression Profile, Promotes Persistent DNA Hypomethylation in Human Brain Endothelial Cells and Impairs Late Cortical Angiogenesis

TL;DR

Ethanol exposure during pregnancy disrupts blood-brain barrier development by altering DNA methylation in brain endothelial cells, leading to long-term vascular issues.

Contribution

This study reveals that ethanol-induced DNA methylation changes in endothelial cells persist and impair cortical angiogenesis, suggesting an epigenetic mechanism for fetal alcohol spectrum disorders.

Findings

Ethanol reduces DNA methylation and alters DNMT1/3a/3b expression in human brain endothelial cells.

Ethanol-induced hypomethylation persists even after ethanol withdrawal and affects BBB-related gene promoters.

SAM treatment prevents ethanol-induced DNA methylation changes in vitro, while PAE increases vascular permeability and disrupts Claudin-5 localization in mice.

Abstract

Exposure of the embryonic central nervous system (CNS) to drugs of abuse, such as ethanol, induces severe and persistent damage to neural cells, contributing to the development of fetal alcohol spectrum disorders (FASD). Previously, using a mouse model of FASD, we showed that prenatal alcohol exposure (PAE) directly impairs blood–brain barrier (BBB) development by inducing excessive angiogenesis, altering TJ protein and glucose transporter expression, and modifying the endothelial secretome in the neonatal cerebral cortex. Here, we investigated whether ethanol‐induced effects on endothelial cells involve epigenetic reprogramming, specifically through alterations in DNA methylation profiles. Using human brain microcapillary endothelial cells (HBMECs) treated with ethanol, we observed reduced 5‐methylcytosine (5mC) labeling intensity and DNA methyltransferase (DNMT) activity, accompanied by changes in the levels of DNMT1, DNMT3a, DNMT3b, methyl‐CpG binding protein 2 (MeCP2), and vascular endothelial zinc finger 1 (VEZF1). These effects were associated with altered methylation levels at the promoters of BBB‐related genes, including GLUT1 and CLDN5. Notably, ethanol‐induced hypomethylation persisted over a prolonged period, even after ethanol withdrawal in HBMEC cultures. Treatment with S‐adenosylmethionine (SAM) prevented ethanol‐induced hypomethylation in vitro. In vivo, PAE resulted in increased cortical vascular permeability along with persistent vascularization deficits. Together, our findings suggest that ethanol induces long‐lasting changes in endothelial cells that may compromise cerebral vasculature formation and function, with modulation of DNA methylation representing a potential molecular mechanism underlying these effects.

Ethanol exposure alters brain endothelial cell function and blood–brain barrier integrity. In human brain microcapillary endothelial cells (HBMEC) and mouse brain endothelial cells (MBEC) from a prenatal alcohol exposure (PAE) model, ethanol increased DNA methyltransferas‐1 (DNMT1) activity and reduced DNA methylation, affecting DNMT3a/3b, methyl‐CpG binding protein 2 (MeCP2) and vascular endothelial zinc finger 1 (VEZF‐1) expression. Epigenetic changes modified promoter methylation of Glut‐1 and Claudin‐5 genes. PAE increased cortical vascular permeability, altered Claudin‐5 mRNA and disrupted Claudin‐5 protein localization at P0. At P22, vascular development presented reduced cortical vascular arborization. SAM treatment rescued ethanol‐induced DNA methylation changes in HBMEC.

Figures