Figure 3.
TGFβ-1 levels are upregulated in EVs from HIV-infected monocyte–derived macrophages (MDMs) on treatment with cocaine, resulting in enhanced MDM EV–mediated endothelial dysfunction and smooth muscle hyperplasia. (A) EVs isolated from supernatants of Mono-Mac-1–derived macrophages infected with HIVBal for 4 days in the presence of cocaine (HIV-infected and Coc [H + C] macrophages) and absence of cocaine (HIV only) or UI MDMs exposed (Coc) or unexposed to cocaine (Con) were analyzed for active (i) and total (ii) TGFβ-1 levels by using an ELISA. (B) HPMECs were treated with 4 μg of MDM EVs, which was followed by performance of a cell-death ELISA at 24 and 48 hours after EV addition (i) and at 24 hours EV addition in the presence or absence of TGFβ-R1 In. (SB431542) (ii). (C) An MTS assay was performed on HPMECs at 48 or 96 hours after treatment with MDM EVs in the absence of SB431542 (i) or at 48 hours after EV treatment in the presence of SB431542 (ii). (D) HPMECs treated with H + C MDM EVs in the presence and absence of SB431542 were lysed by using radio-immunoprecipitation assay buffer at 16 hours after treatment, which was followed by Western blot analysis for phosphorylated and total SMAD2. The graphs represent the densitometry analysis of three independent experiments (mean ± SEM). (E) An MTS assay was performed on HPASMCs treated with H + C MDM EVs in the presence and absence of SB431542 at 48 hours after treatment. (F) Western blot analysis of HPASMCs for phosphorylated and total SMAD2 at 1 hour after treatment. For all In. studies, cells were pretreated with SB431542 (10μM) for 30 minutes before the addition of MDM EVs to cells. Values of experiments using MDM EVs are the average of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 versus Con EVs; ##P < 0.01 and ###P < 0.001 versus Coc EVs; $$P < 0.01 and $$$P < 0.001 versus HIV EVs; and @P < 0.05, @@P < 0.01, and @@@P < 0.001 versus H + C EVs without TGFβ-R1 In. Con = control.