Abstract
BackgroundThere are sex differences in risk for stroke and small vessel ischemic disease in the brain. Microvesicles (MV) derived from activated cells vary by cell of origin and the stimulus initiating their release. MV released from cells activated by inflammatory and thrombotic factors have the potential to disrupt endothelial cells of the brain microvasculature. Therefore, experiments were designed to identify sex differences in the phenotype of MV released from cultured human brain microvascular endothelial cells (HBMEC) in response to inflammatory and thrombotic stimuli.MethodsCultured HBMEC derived from 20- to 30-year-old male and female donors were treated for 20 h with medium supplemented with tumor necrosis factor alpha (TNFα; 20 ng/ml), thrombin (THR; 2 U/ml), or vehicle (i.e., control). MV were isolated from the conditioned media by high-speed centrifugation and quantified by digital flow cytometry by labeling with fluorophore-conjugated primary antibodies against PECAM-1, integrin αvβ3, ICAM-1, E-selectin, or MCAM. In addition, temporal uptake of labeled MV into control HBMEC was examined by confocal microscopy.ResultsUnder control conditions, male HBMEC released fewer MV expressing each antigen, except for PECAM-1, than female cells (P < 0.05). Neither TNFα nor THR reduced cell viability. However, TNFα induced apoptosis in female and male cells, whereas THR increased apoptosis marginally only in male cells. TNFα increased expression of all antigens tested on MV in male cells, but only increased expression of integrin αvβ3, ICAM-1, and E-selectin on MV from female cells. THR increased expression of PECAM-1, ICAM-1, and MCAM-1 on MV from male but not female cells. MV were internalized and localized to lysosomes within 90 min after their application to HBMEC.ConclusionsThere are sex differences in expression of cell adhesion molecules on MV released from HBMEC under control conditions and upon activation by TNFα or THR. MV taken up by unstimulated HBMEC may impact the integrity of the brain microvasculature and account, in part, for sex differences in vascular pathologies in the brain.
Highlights
There are sex differences in risk for stroke and small vessel ischemic disease in the brain
Fluorescein isothiocyanate- conjugated annexin V and phycoerythrin (PE)conjugated antibodies against platelet endothelial cell adhesion molecule-1 (PECAM-1), integrin αvβ3, intercellular adhesion molecule-1 (ICAM-1), E-selectin, and melanoma cell adhesion molecule/cell surface glycoprotein MUC18 (MCAM) for digital flow cytometric analysis (FACS) were obtained from BD Biosciences, San Jose, CA, USA, and CellTiter96® cytotoxicity assay was from Promega, Madison, WI, USA
The total number of each MV phenotype under control conditions ranged from 4.0 ± 1.0 to 15.5 ± 1.1 × 103 in media conditioned by male cells and from 12.4 ± 2.9 to 73.1 ± 6.9 × 103 in media conditioned by female cells
Summary
There are sex differences in risk for stroke and small vessel ischemic disease in the brain. There are sex differences in outcomes from various pathologies involving the cerebral vasculature [6,7,8,9,10] In the brain, these vascular pathological conditions are closely associated with several endothelial processes including release of endothelium-derived microvesicles (MV) [3, 11]. Platelet endothelial cell adhesion molecule-1 (PECAM-1), integrin αvβ, intercellular adhesion molecule-1 (ICAM-1), E-selectin, and melanoma cell adhesion molecule/cell surface glycoprotein MUC18 (MCAM) are upregulated in human cerebrovascular endothelial cells under inflammatory conditions and mediate a multi-step process resulting in transmigration of immune cells into the brain [16,17,18,19,20]. Integrin avβ anchors endothelial cells to matrix components, and to other cell types, and mediates cytoskeletal organization of cells of the blood-brain barrier [17]
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