Ethanol Alters <scp>DNMT</scp> 1/3a/3b Expression Profile, Promotes Persistent <scp>DNA</scp> Hypomethylation in Human Brain Endothelial Cells and Impairs Late Cortical Angiogenesis

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.