Title

MCP-1/CCL2 regulation of brain endothelial tight and adherens junctions

Date of Completion

January 2005

Keywords

Health Sciences, Pharmacology|Health Sciences, Immunology

Degree

Ph.D.

Abstract

A readily obtainable in vitro paradigm of the blood-brain barrier (BBB) would offer considerable benefits. Towards this end, we described a novel method for purifying murine brain microvascular endothelial cells (BMEC) for culture. The method utilized limited collagenase/dispase digestion of enriched brain microvessels, followed by immunoisolation of digested, microvascular fragments by magnetic beads coated with antibody to PECAM-1. When plated onto collagen IV-coated surfaces, these fragments elaborated confluent monolayers of BMEC that retained expression and subcellular localization of the adherens junction (AJ) -associated proteins VE-cadherin and β-catenin, as well as the tight junction (TJ) -associated proteins claudin-5, occludin and zonula occludin-1 (ZO-1). High expression of TJs is considered a seminal characteristic of the BBB, these results impart that this method of purifying murine BMEC provides a suitable platform to investigate BBB properties in vitro . ^ The chemokine monocyte chemoattractant protein (MCP-1) is recognized to mediate extravasation of mononuclear leukocytes into the brain during a variety of neuroinflammatory conditions. In large part produced by parenchymal neural cells during these disease states, it is unclear how this chemokine can stimulate the migration of circulating leukocytes that lie behind the highly impermeant BBB. Based on the premise that disruption of TJ could foster leukocyte extravasation, experiments were conducted to test the hypothesis that MCP-1 has effects on the TJ-associated proteins. Exposure to MCP-1 caused a loss of ZO-1 and occludin protein in BMEC. Moreover, ZO-1 was found reduced in the cytoskeletal framework (CSK) fraction with a shift to the detergent-soluble fraction. In freshly isolated murine brain microvessels, ZO-1 exhibited a similar pattern of MCP-1-induced loss and shift but occludin remained nearly constant. These results indicate that, in addition to its chemotactic activity, MCP-1 might alter BBB integrity during CNS inflammation. ^ To explore the mechanism of MCP-1 induced TJ-associated protein loss, caveolin-1 involved signaling pathway was tested. Coincident with TJ protein alteration, MCP-1 provoked caveolin-1 phosphorylation and a loss of caveolin-1 expression in BMEC. Caveolin-1 knockdown by siRNA adenovirus was sufficient for several TJ and AJ-associated protein down-regulation which accompanied by increased paracellular permeability and monocyte transendothelial migration across BMEC monolayer. These results implicate that caveolin-1 plays an important role in regulation of BBB function. ^