Abstract 3754: Homocysteine Induces Alternations Of Tight Junction Proteins In Brain Endothelial Cells.

Abstract

Hyperhomocysteinmia (HHcy) is associated with neurological disorders (Stroke, Alzheimer, Parkinson etc) and causes blood brain barrier (BBB) dysfunction. We previously showed that an elevated level of homocysteine (Hcy) increased formation of filamentous actin and enhanced endothelial layer permeability. In the present work we tested the hypothesis that Hcy induces oxidative stress and binding to endothelial cells (ECs) alters expression of endothelial tight junction proteins (TJP). In this study mouse brain microvascular endothelial cells (bEND3) were grown in gold plated chambers of an electrical cell-substrate impedance system, 8-well chambered. Confluent bEND3 were treated with different doses of Hcy with mitogen-activated protein kinase (MEK) kinase inhibitors (PD98059 or U0126) or H 2 O 2 (oxidant), or medium alone for 24 h. Reactive oxygen species (ROS) was detected using DCFH-DA assay. Hcy induced a dose-dependent decrease in EC junction integrity as determined by transendothelial electrical resistance (TEER). Our results show that high dose of Hcy induces oxidative stress, which cause down regulation of the TJPs contents occludin, zona occluden-1 (ZO-1), and zona occluden-2 (ZO-2) in bEND3s. Hcy-induced decreases in contents of the TJPs were blocked by PD98059, U0126. While BQ788 inhibited endothelin-1-induced decrease in TEER, it did not affect Hcy-induced decrease in TEER. These data suggest that Hcy increases EC layer permeability via the MEK kinase signaling pathway by affecting TJPs, which are bound to actin filaments. Therefore, increased binding of Hcy with ECs during cerebo-vascular diseases may increase microvascular permeability by altering the content and possibly subcellular localization of endothelial TJPs.