Fig. 3

Exosomal ADAM17 induces vascular endothelial barrier damage by targeting VE-cadherin. (a) Heatmap showing the expression change of proteins in exosomes secreted by Nor-HCT116 and EMT-HCT116 cells based on human serum exosome data. (b) Western blot analysis of ADAM17 and the exosomal marker CD63. (c) Correlation analysis of Exo-ADAM17 protein levels and the protein levels of VE-cadherin, zo-1 and occludin on treated human umbilical vein endothelial cell (HUVEC) membranes. (d, e) HUVECs were incubated with exosomes derived from EMT HCT116 cells (Si-NC and Si-ADAM17 transfected). Dextran was detected (OD 590 nm) in the lower chamber and the number of tumor cells invading the HUVEC monolayer was determined. (f, g) HUVECs were incubated with exosomes derived from EMT HCT116 cells (Si-NC and Si-ADAM17 transfected); TEER and CI values were calculated. (h–j) HUVECs were incubated with exosomes derived from EMT HCT116 cells (Si-NC and Si-ADAM17 transfected); cell membrane VE-cadherin levels were measured using western blotting, flow cytometry, and immunofluorescence (scale bar = 25 μm). Data are expressed as the mean ± standard deviation (n = 5). **P < 0.01, compared with the control group; #P < 0.05, ##P < 0.01, compared with the Si-NC group. (k) Exosomes were pre-incubated with ADAM17 selective inhibitor-JG26 (25 nM), ADAM17 oral inhibitor aderbasib (1 µM), and ADAM10 selective inhibitor-GI254023 × (25 nM) for 2 h before culturing with HUVECs. (l, m) Dextran content was detected (OD 590 nm) in the lower chamber, and the number of tumor cells invading the HUVEC monolayer was determined. (n, o) TEER and CI values. (p–r) Cell membrane VE-cadherin levels were determined using western blotting, flow cytometry, and immunofluorescence (scale bar = 25 μm). Data are expressed as mean ± standard deviation (n = 5). **P < 0.01, compared with the control group; #P < 0.05, ##P < 0.01, compared with the EMT-HCT116-Exo group