Figure 6.

Blocking integrin αvβ1 attenuates sCD146-induced BBB dysfunction. (A) rhsCD146-induced paracellular permeability of hCMEC/D₃ cells was blocked by anti-integrin αv and β1 antibodies. hCMEC/D₃ cells were seeded to the upper chambers of a transwell system; preincubated with 3 μg/mL IgG, anti-integrin αv or β1 antibody for 30 min; and then cultured with 5 μg/mL BSA or rhsCD146 for another 2 h. Permeability was measured using 0.5 μg/mL HRP. *p<0.05; **p<0.01; and ***p<0.001. (B) hCMEC/D₃ cells were preincubated with 3 μg/mL IgG, anti-integrin αv or β1 antibody for 30 min and then treated with 5 μg/mL BSA or rhsCD146. TJP expression was verified by western blotting. (C) Immunofluorescence staining of the TJPs and F-actin in hCMEC/D₃ cells pretreated with 3 μg/mL IgG, anti-integrin αv or β1 antibody for 30 min and 5 μg/mL BSA or rhsCD146 for 4 h. Bar, 10 μm. (D) hCMEC/D₃ cells were preincubated with 3 μg/mL IgG, anti-integrin αv or β1 antibody for 30 min and treated with 5 μg/mL BSA or rhsCD146 for another 12 h; apoptosis was detected by flow cytometry with Annexin V and 7-AAD. (E-F) hCMEC/D₃ cells were preincubated with 3 μg/mL IgG, anti-integrin αv or β1 antibody for 30 min and treated with 5 μg/mL BSA or rhsCD146 for another 12 h; western blotting was performed to detect the expression of caspase 9, caspase 3, Bcl-2 and Bax.