Fig. 2

NRG-1 attenuates heme-induced damage of human brain microvascular endothelial cells (hCMEC/D3). a–d A monolayer of hCMEC/D3 cells were grown confluently and treated with 30 μM heme and NRG-1100 ng/ml followed by staining with anti-claudin5 antibody against tight junction protein claudin5. Claudin5 was expressed on cell membranes (arrows) which disappeared after the cells were treated with 30 μM heme for 24 h. NRG-1 treatment inhibited the disruption of heme-induced Claudin5 membrane expression. e Claudin5 antibody immunoreactive signals in hCMEC/D3 cells were quantitatively analyzed under each treatment as indicated. The fluorescence intensity (red) of cells was measured in 500 cells per treatment using an ImageJ software program (Version 1.51j8, National Institutes of Health (NIH)). The average relative intensity of cells associated with claudin5 antibody was 177.44 ± 16.45 in controls. Heme treatment reduced the average intensity to 129.33 ± 13.16 (p < 0.05). In contrast, NRG-1 treatment increased the average fluorescence intensity back to 165.23 ± 17.11 (p < 0.05). The asterisks indicate significant difference with a p value of 0.05 as determined with Student’s paired t test. f–i hCMEC/D3 cells were grown in monolayer treated with 30 μM heme and 100 ng/ml NRG-1 and stained with anti ZO-1 antibody against tight junction protein ZO-1. ZO-1 is expressed on cell membranes (arrows) and disappeared after cells were treated with 30 μM heme for 24 h. NRG-1 treatment inhibited disruption of heme-induced ZO-1 membrane expression. j Quantitation of ZO-1 staining from samples of hCMEC/D3 cells treated with heme, NRG-1, combination of heme and NRG-1, and corresponding control. Total immunofluorescence intensity with ZO-1 antibody (red) was determined with ImageJ software program, and the average values for the indicated number of cells (n = 500) examined were as shown. Briefly, the average relative intensity of ZO-1 of heme-treated cells decreased to 96.99 ± 10.35 as compared to controls 136.16 ± 13.00 (p < 0.05). The average immunofluorescence intensity with ZO-1 antibody increased to 125.45 ± 11.78 when NRG-1 was added to heme-treated cells (p < 0.05). The asterisks indicate significant difference with a p value of 0.05 as determined with Student’s paired t test. k–n hCMEC/D3 cells were grown in monolayer treated with 30 μM of heme and NRG-1100 ng/ml and thereafter stained with anti-occludin antibody. Occludin is expressed on cell membranes (arrows) and disappeared after cells were treated with 30 μM heme for 24 h. NRG-1 treatment prevented the disruption of heme-induced occludin expression. o The average relative intensity of occludin in heme-treated cells decreased to 88.04 ± 9.24 compared to controls 137.34 ± 14.23 (p < 0.05), while the average immunofluorescence intensity increased to 136.00 ± 15.00 when NRG-1 was added to heme-treated cells (p < 0.05). The asterisks indicate significant difference with a p value of 0.05 as determined with Student’s paired t test