Figure 2.

TGF-β negatively modulates the integrity of the BBB in vitro. (A) TGF-β negatively modulates the integrity of the barrier. Addition of TGF-β1 plus -β2 (each 5 ng/mL; grey bar) into the bottom chamber (HBVP cell membrane side) of barrier-tight BBB layers. Twenty-four hours later, the integrity of the barrier was determined by TEER (Co: sham treatment; black bar). n = 4, each 2 technical replicates, SEM, t-test, ** p < 0.01). (B) The cells were treated with TGF-β1 plus -β2 [each 3 ng/mL (3+3) or 6 ng/mL (6+6)] in the absence or presence of the pan-TGF-β neutralizing antibody 1D11 (1 µg/mL). Addition of 1D11 reverted the TGF-β mediated reduction in BBB integrity (Co: sham treatment, n = 2, each one replicate, SD). (C) Intact barrier-dense BBB layers were transferred into new wells containing either no cells (black bars) or T98G cells (green bars) as well as no antibody (-), 1D11 antibody, or an isotype control (IgG, each 1 µg/mL). Twenty-four hours later, TEER measurement was performed (n = 3, each 2 replicates, SEM, t-test). (A–C) Circles, triangles and squares at the appropriate bars present the data points of single experiments. (D) PBMVEC monoculture and PMBVEC/HBVP co-culture membranes were treated with TGF-β1 plus -β2 (each 5 ng/mL) 48 h after seeding. TEER measurement was performed as described in the Methods section (one out of three independent experiments is shown, light-colored lines indicate the SD of technical replicates). (E) Differences in the reduction in TEER in PBMVEC mono- and PBMVEC/HBVP co-cultures after addition of TGF-β1 plus -β2 (each 5 ng/mL; n = 3, SD, p = 0.18).