Endothelial-Derived Extracellular Vesicles Induce Cerebrovascular Dysfunction in Inflammation.

David Roig-Carles,Sarai Martinez-Pacheco,Imre Mäger,Eduard Willms,Ignacio A Romero,David K Male,Cheryl A Hawkes,Helga E De Vries,Ruud D Fontijn,Basil Sharrack,Mark Hirst

doi:10.3390/pharmaceutics13091525

Abstract

Blood–brain barrier (BBB) dysfunction is a key hallmark in the pathology of many neuroinflammatory disorders. Extracellular vesicles (EVs) are lipid membrane-enclosed carriers of molecular cargo that are involved in cell-to-cell communication. Circulating endothelial EVs are increased in the plasma of patients with neurological disorders, and immune cell-derived EVs are known to modulate cerebrovascular functions. However, little is known about whether brain endothelial cell (BEC)-derived EVs themselves contribute to BBB dysfunction. Human cerebral microvascular cells (hCMEC/D3) were treated with TNFα and IFNy, and the EVs were isolated and characterised. The effect of EVs on BBB transendothelial resistance (TEER) and leukocyte adhesion in hCMEC/D3 cells was measured by electric substrate cell-substrate impedance sensing and the flow-based T-cell adhesion assay. EV-induced molecular changes in recipient hCMEC/D3 cells were analysed by RT-qPCR and Western blotting. A stimulation of naïve hCMEC/D3 cells with small EVs (sEVs) reduced the TEER and increased the shear-resistant T-cell adhesion. The levels of microRNA-155, VCAM1 and ICAM1 were increased in sEV-treated hCMEC/D3 cells. Blocking the expression of VCAM1, but not of ICAM1, prevented sEV-mediated T-cell adhesion to brain endothelia. These results suggest that sEVs derived from inflamed BECs promote cerebrovascular dysfunction. These findings may provide new insights into the mechanisms involving neuroinflammatory disorders.

Highlights

We found that the cytokine challenge of hCMEC/D3 cells induced an increased release of small EVs (sEVs) that contained proinflammatory modulators
Supplemented with 0.025% (v/v) recombinant human epithelial growth factor, 0.025% (v/v) vascular endothelial growth factor (VEGF), 0.025% (v/v) insulin growth factor (IGF), 0.1% (v/v) recombinant human fibroblast growth factor, 0.1% (v/v) gentamycin, 0.1% (v/v) ascorbic acid, 0.04% (v/v) hydrocortisone and 2.5% (v/v) foetal bovine serum (FBS) (Lonza, Wilford, Nottinghamshire, UK), hereafter referred to as the endothelial complete medium. hCMEC/D3 cells were used from passages 25–35 and grown in calf skin collagen-coated tissue culture flasks until confluence unless otherwise indicated
We investigated the relative levels of mRNAs that are directly involved in T-cell adhesionand andobserved observed that was significantly increased involved in T-cell adhesion thatICAM1

Summary

Introduction

BECs are the main cellular component of the BBB, a specialised feature of the vasculature of the central nervous system (CNS) [1]. BEC dysfunction is common in neuroinflammatory pathologies like multiple sclerosis (MS) [2]. Inflammatory modulators such as proinflammatory cytokines are upregulated in the plasma of individuals with MS and other conditions such as sepsis [3,4]. Cytokines play an additional central role in the progression of inflammation by acting on the BBB. Tumour necrosis factor alpha (TNFα) and interferon gamma (IFNy) modulate the vascular function by increasing the paracellular

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