Fig. 4.

TF-bearing MVs trigger p-ERK1/2 and HB-EGF and colocalize with PAR-2 in hypoxic ECs. (A–D) MVs from hypoxic GBM cells trigger p-ERK1/2 and HB-EGF in hypoxic ECs in a TF-dependent manner. (A) MVs isolated from hypoxic U87-MG cells were incubated with HUVECs at the indicated concentrations for 5 min, followed by immunoblotting for p-ERK1/2, total ERK1/2, and α-tubulin. Lower: p-ERK/α-tubulin ratios from a representative experiment. (B) Hypoxic HUVECs were untreated or incubated with MVs (10 μg/mL) from hypoxic U87-MG cells for 5 min in the absence or presence of increasing concentrations of NAPc2 as indicated. Shown are immunoblots for p-ERK1/2, total ERK1/2, and β-actin. Lower: p-ERK/β-actin ratios from a representative experiment. (C) Hypoxic HUVECs were untreated or treated with MV as in B in the absence or presence of anti-human TF antibody (α-TF; 1:100), followed by immunoblotting for p-ERK1/2, total ERK1/2, and α-tubulin. Lower: p-ERK/α-tubulin ratios from a representative experiment. (D) Hypoxic HUVECs were incubated without or with MVs (10 μg/mL) for 1 h in the absence or presence of α-TF (1:100); HB-EGF mRNA expression was determined by qRT-PCR. (*P < 0.05.) (E–H) Vesicular transfer of TF from GBM cells into a PAR-2–containing compartment of ECs. (E) Confocal microscopy of U87-MG cells and HUVECs cultured in hypoxia for 24 h in the presence of the EC marker Ac-LDL (red). Cells were fixed and stained for TF (green) and nuclei (blue). HUVECs were positive for Ac-LDL and negative for TF, whereas the reverse was found in U87-MG cells. Right: Coincubation experiment with U87-MG cells and HUVECs in hypoxia for 24 h. Detail shows representative HUVEC containing TF-positive vesicular structures. (F) MVs (40 μg/mL) isolated from hypoxic U87-MG cells were incubated with hypoxic HUVECs for 30 min, and ECs were analyzed by Immunogold labeling for TF and EM as described in Materials and Methods. Shown is a TF-positive MV at the surface of ECs. (G) Hypoxic HUVECs were incubated without (Ctl) or with MVs from hypoxic U87-MG cells for 30 min, and then analyzed by Immunogold labeling for TF (5-nm gold particle, arrow) and PAR-2 (15-nm gold particle, arrowhead), followed by EM analysis. Upper: PAR-2 staining at the EC surface. Lower: Localization of TF-labeled MVs in a PAR-2–positive compartment of ECs. (H) MVs (10 μg/mL) time-dependently induce PAR-2 internalization in hypoxic HUVECs, as determined by EC surface staining for PAR-2 and flow cytometry. Data are presented as average ± SD. (*P < 0.05.)