Figure 3.

Pericyte coverage, tight junctions and endothelial leakiness during acute graft-versus-host disease. Organs were harvested at day+15 after experimental hematopoietic stem cell transplantation (HSCT) in the chemotherapy based LP/J→B6 model. Control groups (no acute graft-versus-host disease [aGvHD]) were transplanted with the same bone marrow cell numbers and T-cell numbers from syngeneic donors. (A, B, and G to H) Quantification of pericyte coverage of vessels. (A and G) Representative pictures of staining for pericyte marker α smooth muscle actin (αSMA) in red and endothelial cell marker CD31 in green. Right organs of animals without aGvHD and left organs of animals suffering from aGvHD. (B and H) Ratio of αSMA positive area and CD31 positive area in (B) liver sinusoidal endothelium and (H) in colonic mucosal vessels in aGvHD versus no aGvHD. (C, D, I and J) Quantification of endothelial tight junction protein expression ZO-1. (C and I) Representative pictures of staining ZO-1 in green and CD31 in red. Right organs of animals without aGvHD and left organs of animals suffering from aGvHD. (D and J) Percentage of ZO- 1⁺ CD31⁺ area in (D) liver sinusoidal endothelium and (J) colonic mucosal vessels in aGvHD versus no aGvHD. (E and K) Quantification of endothelial adherence junction protein expression VE-cadherin. Percentage of VE-cadherin positive area in (E) liver sinusoidal endothelium and (K) colonic mucosal vessels in aGvHD versus no aGvHD. (F and L) Measurement of Evans blue extravasation in ng Evans blue per mg at day+15 after experimental allo-HSCT in the chemotherapy based B6→BDF model. (F) Liver and (L) colon of aGvHD versus no aGvHD. Significance was tested with Student’s t-test (*P<0.05; **P<0.01; n=5 animals per group). All experiments were reproduced in a biological independent experiment and shown are representative results of one experiment. Error bars indicate mean ± standard error of the mean.