Elsevier

Brain Research

Volume 1513, 4 June 2013, Pages 1-8
Brain Research

Research Report
Involvement of PI3K and ROCK signaling pathways in migration of bone marrow-derived mesenchymal stem cells through human brain microvascular endothelial cell monolayers

https://doi.org/10.1016/j.brainres.2013.03.035Get rights and content

Highlights

  • MSC-induced tight junction disassembly in brain microvascular endothelial cells.

  • PI3K and ROCK signaling were involved in the MSC-induced increase in endothelial cell permeability.

  • Dominant negative PI3K mutations in MSC inhibited MSC transendothelial migration.

  • Interference with ROCK in MSC enhanced MSC transendothelial migration.

Abstract

Bone marrow-derived mesenchymal stem cells (MSC) represent an important and easily available source of stem cells for potential therapeutic use in neurological diseases. The entry of circulating cells into the central nervous system by intravenous administration requires, firstly, the passage of the cells across the blood–brain barrier (BBB). However, little is known of the details of MSC transmigration across the BBB. In the present study, we employed an in vitro BBB model constructed using a human brain microvascular endothelial cell monolayer to study the mechanism underlying MSC transendothelial migration. Transmigration assays, transendothelial electrical resistance (TEER) and horseradish peroxidase (HRP) flux assays showed that MSC could transmigrate through human brain microvascular endothelial cell monolayers by a paracellular pathway. Cell fractionation and immunofluorescence assays confirmed the disruption of tight junctions. Inhibition assays showed that a Rho-kinase (ROCK) inhibitor (Y27632) effectively promoted MSC transendothelial migration; conversely, a PI3K inhibitor (LY294002) blocked MSC transendothelial migration. Interestingly, adenovirus-mediated interference with ROCK in MSC significantly increased MSC transendothelial migration, and overexpression of a PI3K dominant negative mutant in MSC cells could block transendothelial migration. Our findings provide clear evidence that the PI3K and ROCK pathways are involved in MSC migration through human brain microvascular endothelial cell monolayers. The information yielded by this study may be helpful in constructing gene-modified mesenchymal stem cells that are able to penetrate the BBB effectively for cell therapy.

Graphical abstract

In vitro studies of MSC transendothelial migration revealed that MSC transendothelial migration was associated with the PI3K and ROCK pathways. Interestingly, a ROCK inhibitor (Y27632) or adenovirus-mediated interference with ROCK expression in MSC significantly increased MSC transendothelial migration. These results indicate that genetically modified mesenchymal stem cells that are able to penetrate the BBB can be constructed for cell therapy of CNS disease.

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Introduction

Bone marrow-derived mesenchymal stem cells (MSC) have the potential to differentiate into mesenchymal lineage cells (Makino et al., 1999, Matsushita et al., 2011); additionally, they can be experimentally induced to undergo unorthodox differentiation into neural and myogenic cells (Bianco et al., 2001). As such, MSC represent an important paradigm and an easily available source for potential therapeutic use in neurological diseases (Lee et al., 2010, Danielyan et al., 2009, Inoue et al., 2003, Li et al., 2002). Considering their possible use in cell therapy, injection of cells into the venous blood (Ali et al., 2012) is a routine medical procedure (Sokolova et al., 2007) that is often used because it is less invasive than local administration (Olson et al., 2012).

The primary structural basis of the integrity of the blood–brain barrier (BBB) is the presence of tight junctions between microvascular endothelial cells throughout the brain; these junctions regulate the paracellular passage of cells, molecules, and ions. The entry of circulating cells into the central nervous system (CNS) requires, firstly, the passage of the cells across the vascular endothelial cell layer. It has been reported that peripheral blood T lymphocytes (Man et al., 2007) and small-cell lung cancer cells (Li et al., 2006) can “open” the tight junction of human brain microvascular endothelial cells (HBMEC) and trigger subsequent transendothelial migration. MSCs are capable of transendothelial migration through ES cell-derived endothelial cell monolayers with the disruption of tight junction (Schmidt et al., 2006). However, the defined molecular mechanisms involved in MSC transmigration through the BBB remain unknown.

In the present study, we employed an in vitro BBB model reported previously (Man et al., 2007, Li et al., 2006) to study the mechanism underlying MSC transendothelial migration and found that the phosphatidylinositol 3-kinase (PI3K)/Akt and Rho/ROCK (Rho kinase) pathways of MSC are involved in the transmigration process.

Section snippets

MSC trigger tight junction disassembly in HBMEC monolayers

HBMEC monolayers cultured on Transwell inserts have been used as an in vitro BBB model (Man et al., 2007, Li et al., 2006) to study the mechanism of transendothelial migration in cells in suspension by measuring the cells that crawl through the membrane to the lower chamber. However, adherent MSC attached to the bottom of the porous membrane instead of remaining in the medium in the lower chamber. By referring to the adherent breast cancer cell migration model (Lee et al., 2004), we developed

Discussion

According to Dharmasaroja, the therapeutic potential of MSC in various pathological conditions of the CNS has been explored (Dharmasaroja, 2009). Although transplantation of MSC through intravenous injection represents a minimally invasive strategy (Li et al., 2002), the delivery of cells into the brain is limited due to the existence of the BBB. A better understanding of the mechanisms underlying MSC transendothelial migration will be helpful in designing successful cellular therapies for CNS

Cell culture

Rat MSC were prepared as described by Tropel et al. (2004). Briefly, male Wistar rats were sacrificed by cervical dislocation; the femurs were aseptically dissected, repeatedly flushed with MSC medium containing DMEM-LG, 10% fetal bovine serum (FBS, Hyclone) and 2 mM glutamine, and plated in a culture flask (Corning Costar). Twenty-four hours later, the non-adherent cells were removed, and the adherent cells were cultured for 14 days as passage 0. MSC at passages 3–8 were used in the

Acknowledgments

The authors are grateful to Drs. Monique Stins and Kwang Sik Kim (Department of Pediatrics, John Hopkins University School of Medicine) for providing HBMEC. This work was supported by the Innovation Team Program Foundation of Liaoning Province (2006T131, LT2011011), Liaoning Province Doctoral Startup Fund (20061035), National Natural Science Foundation of China (30970120, 31171291) and National Program on Key Basic Research Project (2013CB531003).

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    These authors contributed equally to this work.

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