Fig. 3

BMEC EVs and their effect on immune cell migration in the context of HIV. a Analysis of TJ proteins and endothelial markers in MVs released by BMECs following HIV virotoxin exposure. HIV virotoxins, gp120MN and TatYU2 induce increased levels of TJP release on MVs and of transporter protein p-glycoprotein, Pgp. b Representative image of immunofluorescence showing colocalization of TJP occludin with monocytic marker after incubation of monocytes with BMEC-EVs (right). To demonstrate exogenous occludin expression due to EV interaction/uptake, monocytes were incubated with EVs isolated from BMECs transfected with occludin-GFP (left). Scale bar = 10 microns. c Monocytes exhibit increased transendothelial migration when exposed to BMEC derived EVs. Migration assays were conducted for 4 h during which fluorescently labeled monocytes w/o EVs were introduced to BMECs grown on fluoroBlok inserts (Corning©). Monocytes were isolated from blood filters using a PAN-monocyte isolate kit (Miltenyi Biotech). EVs were isolated from BMECs treated for 24 h with 50 ng/ml of HIV-1 Tat (YU2). Isolated EVs were incubated with 1x10⁵ monocytes. MCP-1 at 20 ng/ml was included where indicated to initiate the migration. Results from counts of migrated monocytes are shown as the average + SEM, (*) P < 0.05