Sex differences in the expression of cell adhesion molecules on microvesicles derived from cultured human brain microvascular endothelial cells treated with inflammatory and thrombotic stimuli

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 20h with medium supplemented with tumor necrosis factor alpha (TNF; 20ng/ml), thrombin (THR; 2U/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 v3, 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 TNF nor THR reduced cell viability. However, TNF induced apoptosis in female and male cells, whereas THR increased apoptosis marginally only in male cells. TNF increased expression of all antigens tested on MV in male cells, but only increased expression of integrin v3, 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 90min 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 TNF 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.