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Endothelium‐Derived Microvesicles Alter Permeability of Human Brain Microvascular Endothelial Monolayers
Author(s) -
Hunter Larry W.,
Jayachandran Muthuvel,
Miller Virginia M.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.957.9
Subject(s) - chemistry , endothelium , permeability (electromagnetism) , blood–brain barrier , dextran , tumor necrosis factor alpha , agonist , vascular permeability , inflammation , endothelial stem cell , pharmacology , immunology , biophysics , endocrinology , receptor , medicine , in vitro , biochemistry , biology , central nervous system , membrane
The disruption of the cerebral microvascular endothelium and increases in capillary permeability are characteristic of many brain pathologies, including Alzheimer's disease, small vessel disease, ischemia/hypoxia, and hemorrhagic stroke. This study was designed to investigate whether microvesicles (MVs) released from activated human brain microvascular endothelial cells (HBMECs) alter permeability of endothelial monolayers. Male donor HBMECs were cultured to confluence in flasks, then treated for 24 hrs with medium alone (control) or with medium plus either tumor necrosis factor (TNF a , 20 ng/ml), thrombin (THR, 2 U/ml), angiotensin II (AngII, 10 nM), or hydrogen peroxide (H 2 O 2 , 0.8 mM). MVs released into the medium were isolated by differential centrifugation, labeled with annex in V plus antibodies against endothelial adhesion molecules which have modified expression during inflammation (PECAM‐1, ICAM‐1, E‐select in, integrin aV, or MCAM); and then analyzed by FACS. Total number of MVs released (MVs/ml) by each of the agonists (TNF a , 615.8±13.9 MV/μL; THR, 113.8±4.8 MV/μL; and H 2 O 2, 152.8±23.5 MV/μL) were significantly greater (P<0.05) compared the number released from cells treated medium alone (82.2±5.4 MV/μL). Each agonist treatment released distinct types of MV identified by FACS. Permeability (% control) of the HBMEC monolayer assessed using FITC‐dextran transport across the monolayers increased after 90 minute exposure to all agonist treatments: 199.5%, 234.1%, 126.8%, and 588.3% for TNF a , THR, AngII, and H 2 O 2 , respectively. MV derived from the treated cells were labeled with PKH67 and applied to monolayers of (control) HBMECs cultures. Within 30 minutes of exposure punctate structures with PKH67 labeling were observed within the cytoplasm, and after 60 min, similar structures also associated with nuclei and some of which were co‐labeled with the lysosome marker lysotracker red. Co‐localization of labeled MVs with early endosomes, (labeled with anti‐EEA‐1 Ab/Alexa Fluor 647) was absent at these time points. Within these parameters, no differences in vesicle‐trafficking were observed between MVs generated from cells exposed to either medium, TNF a , or H 2 O 2 . These results suggest that treatment of HBMECs with agonists associated with inflammation and thrombosis increase release of distinct MVs which in turn increase paracellular permeability. Capillary endothelial permeability may be disturbed by populations of MVs released from proximal activated cells or MV derived from activated cells and carried in the blood. Support or Funding Information Supported by the Aurora Fnd, NIH HL88988 and Mayo Clinic