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Phenotypic and Sex Differences in Microvesicles Released from Cultured Human Brain Microvascular Endothelial Cells in Response to Proinflammatory and Prothrombotic Stimuli
Author(s) -
Hunter Larry W.,
Jayachandran Muthuvel,
Miller Virginia M.
Publication year - 2017
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.31.1_supplement.684.7
Subject(s) - proinflammatory cytokine , annexin , flow cytometry , tumor necrosis factor alpha , endothelium , microvesicles , thrombin , blood–brain barrier , vascular permeability , endothelial stem cell , chemistry , platelet , microbiology and biotechnology , immunology , inflammation , biology , endocrinology , in vitro , biochemistry , central nervous system , microrna , gene
Objective Activation of brain microvascular endothelium by inflammatory and procoagulant stimuli increases expression of surface cell‐adhesion molecules (CAMs), releases endothelium‐derived microvesicles (MV) into the circulation, and increases permeability of the blood‐brain barrier (BBB). This study evaluated characteristics of MV released from human brain microvascular endothelial cells (HBMEC) following stimulation with proinflammatory or procoagulant stimuli and examined their effect on monolayer permeability. Methods Cultured HBMECs from age‐matched male and female donors were treated for 24 hours with medium supplemented with tumor necrosis factor alpha (TNF α , 20 ng/mL), thrombin (THR, 2 U/mL), or vehicle (control). MVs released into the medium were isolated by differential centrifugation, labeled with annexin V together with antibodies against PECAM‐1, ICAM‐1, E‐selectin, integrin aV, or MCAM, and then analyzed by digital flow cytometry. Permeability (mg/mL FITC‐dextran) of the HBMEC monolayer in response to MV treatment was quantified by fluorescence microplate assay of passage of FITC‐dextran across monolayer cells cultured on microporous supports. Results Fewer specific antigen‐positive MV were released from unstimulated male compared to female HBMECs. TNF a , but not THR induced early‐stage apoptosis; determined by fluorogenic Caspase‐3 assay in the HBMEC monolayers. TNF a induced release of all MV subtypes from male cells, whereas only integrin aV, ICAM‐1 and E‐selectin positive MVs were released from female cells. THR increased release of PECAM‐1, ICAM‐1, and MCAM positive MVs from male cells, but did not increase release of MV in female cells. After 24 hours exposure to MV (2,500 MV/mL) derived from TNF a ‐treated female cells, permeability increased by 17%. Permeabiity was not increased when female cells were exposed to THR‐generated MV. Conclusions These results indicate that: 1) inflammatory and procoagulant stimuli release a different array of MV subtypes from male and female HBMECs; 2) specific stimuli‐derived MVs can modify HMBMEC permeability. Support or Funding Information (Supported by NIH P50 AG44170 and the Mayo Clinic Fnd.).