Fig. 2.

RBCs pass through the damaged endothelium. (A) Confocal images showing changes in the HUVEC monolayer after treatment with APL-CM for 0, 4, 8, and 16 h (upper row). The HUVEC monolayer was pretreated with asiatic acid and induced with APL cell medium (lower row). The monolayer was stained with DAPI (blue) and for VE-cadherin (green) and F-actin (red). Arrows indicate gaps. Images representative of 3 independent experiments are shown. (B) The size distribution of the intercellular gaps induced by different levels of HUVEC stimulation; n = 200 gaps from 10 glass coverslips. (C) Electron micrographs showing different sized gaps (arrows) after 24 h of NB4/APL cell medium treatment. The upper panels show tight connections (left) and a gap of diameter 3.63 µm (right); the lower panels show gaps of 6.01 µm (left) and 14.14 µm (right). (D) Schematic representation of the RBC leakage experiment. (E) The number of leaked RBCs after endothelial monolayer incubation with RBCs. (F) Experimental scheme for RBC deposition. The HUVEC monolayer was pretreated with APL cells for 24 h to induce junction breakage. As the gaps grew in size and number, an increased area was exposed to which the RBCs could adhere. (G) RBCs (red) adhered to openings within endothelial monolayer treated as in (A). Arrows point to the RBCs deposited in the gaps. (H) The number of RBCs adhesion to the gaps at 16 h. Scale bars represent 20 µm in panels A, C, and G. The results indicate the mean ± SD of at least five experiments. *P <0.05 vs. the control group, **P <0.01 vs. the NB4 group, and ^#P <0.05 vs. the APL group in panel B. *P <0.01, **P <0.001 in panel E, *P <0.05 in panel H. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)