Fig. 4.
(A) Zinc metalloprotease activity is required for soluble TNFR1 release. HUVEC were treated with 0.1% DMSO, 25 μM Z-VAD-FMK, or 50 μM TAPI-2 for 24 h, and concentrations of sTNFR1 present in culture supernatants were determined by ELISA (n = 6). *, P < 0.05 vs. DMSO-treated cells. (B) Effect of TAPI-2 on TNFR1 exosome-like vesicle release. HUVEC were treated with (+)(C and D) or without (–) (A and B) 50 μM TAPI-2 for 24 h, and medium was subjected to sequential centrifugation. Samples of proteins (60 μg), in duplicate, from whole cell lysates (T) and 175,000 × g (P6) pellets were separated by SDS/PAGE, transferred to nitrocellulose, and reacted with antibodies against TNFR1. The average relative mean density of the TAPI-2-treated whole cell lysates was 1.56 as compared with 1.0 for the cells treated with medium alone, whereas the relative mean density of the TAPI-2-treated exosomes was 1.0 as compared with 1.75 for exosomes treated with medium alone. (C) HUVEC-derived exosomes bind TNF-α. Exosomes were isolated from HUVEC medium as described, resuspended in medium, and incubated with 1 ng of recombinant human TNF-α for 16 h at 4°C. Exosomes were recovered by centrifugation at 175,000 × g for 16 h at 4°C, separated by SDS/PAGE, transferred to nitrocellulose, and reacted with antibodies against TNF-α. (D) Binding of rhTNF-α to HUVEC-derived exosomes. TNFR1 exosomes (100 μg) were immunoprecipitated with a murine monoclonal antibody directed against the TNFR1 extracellular domain (clone 16805.21) and incubated with rhTNF-α, and the quantity of bound rhTNF-α, in duplicate samples, was measured by ELISA.