Fig 2: TRAF4-NOX complex induced ICAM1 expression through the NF-?B pathway in response to radiation. (A) The effects of knockdown of TRAF4, NOX2, or NOX4 on activation of NF-?B signaling were detected by Western blotting. Cell lysates were fractionated into cytosolic extract (CE) and nuclear extract (NE). Tubulin and Lamin A/C were used as markers for CE and NE, respectively. (B) Effects of knockdown of TRAF4, NOX2, or NOX4 on transcriptional activity of NF-?B were measured by a luciferase assay. Error bars, ± SD (n = 3); * p < 0.05 compared with non-transfected and non-irradiated cells, ** p < 0.05 compared with irradiated cells. (C) Effects of knockdown of TRAF4, NOX2, or NOX4 on radiation-induced mRNA expression of ICAM1 were assessed by qRT-PCR. Error bars, ± SEM (n = 3); * p < 0.05 compared with non-irradiated cells, ** p < 0.05 compared with irradiated cells. (D) Effects of knockdown of TRAF4, NOX2, or NOX4 on secretion of sICAM1 were measured by ELISA in CM of normal lung fibroblasts. Error bars, ± SEM (n = 3); * p < 0.05 compared with non-irradiated cells, ** p < 0.05 compared with irradiated cells.

Fig 3: ICAM-1 activates the caspase-3 enzyme. A–C, AP immunohistochemistry of cl-caspase-3 in the PFC (A) and hippocampus (C) tissue sections from WT and ICAM-1-/- mice 48 h after 10 and 20 psi FPI using vector purple substrate kit, SK4600 in A and vector blue substrate kit, SK5300 in C. Scale bar: 100 µm (A, black), 20 µm (A, blue), and 100 µm (C, yellow). B, D, Quantification of cl-caspase-3-positive cells expressed as per mm2 area of sections in WT and ICAM-1-/- in uninjured, 10 and 20 psi groups in the PFC (B) and hippocampus (D) 48 h following FPI (n = 6/group). E, F, Western blotting of cl-caspase-3 and ß-actin in the PFC (E) and hippocampus (F) tissue lysates of WT and ICAM-1-/- mice 48 h after 10 and 20 psi FPI. The bar graph shows the quantification of cl-caspase-3 versus ß-actin (n = 6/group). G, Western blotting of cl-caspase-3 and ß-actin 24 h after 3.0 psi stretch injury in the cell lysates of hBMVEC treated with control siRNA, ICAM-1 siRNA, A205804, and IRP (n = 6/group). Bar graph represents the densitometric ratio of cl-caspase-3 bands versus ß-actin bands. All values are expressed as mean ± SD one-way ANOVA for G and two-way ANOVA for B, D–F followed by Bonferroni post hoc tests. Statistically significant ***p < 0.001 versus WT uninjured group in B, D, E, F; @p < 0.05; @@p < 0.01, @@@p < 0.001 versus uninjured ICAM-1-/- group in B, D, E, F; ###p < 0.001 versus corresponding WT uninjured or injury groups (10 and 20 psi) in B, D, E, F; ***p < 0.001 versus uninjured hBMVEC in G; #p < 0.05, ###p < 0.001 versus 3 psi injury in G; ns = non-significant.

Fig 4: Stretch injury causes the activation of ICAM-1 in a time-dependent manner and is regulated by oxidative stress, MMPs, and VEGF signaling. A, Western blot analysis of ICAM-1 and ß-actin 24 h after 3.0 psi stretch injury in the cell lysates of hBMVEC treated with control siRNA, ICAM-1 siRNA, A205804, and IRP. Bar graph represents the quantification of ICAM-1 versus ß-actin (n = 6/group). B, C, ELISA quantification of ICAM-1 in cell lysates (B), and cell culture supernatant (C) of hBMVEC following 3.0 psi stretch injury treated with control siRNA, ICAM-1 siRNA, A205804, and IRP (n = 6/group). D, mRNA expression level of ICAM-1 using qPCR from hBMVEC treated with control siRNA, ICAM-1 siRNA, A205804, and IRP (n = 6/group) 24 h after 3.0 psi stretch injury. E, Western blot analysis of ICAM-1 and ß-actin expression at different time points (1, 6, 12, 24, 48 h) in hBMVEC lysates after 24 h 3.0 psi stretch injury. Bar graph represents the quantification of ICAM-1 versus ß-actin (n = 6/group). F, G, ELISA quantification of ICAM-1 in cell lysate (F), and cell culture supernatant (G) of hBMVEC at different time points (1, 6, 12, 24, 48 h) following 3.0 psi stretch injury. H, Western blot analysis of ICAM-1 and ß-actin 24 h after 3.0 psi stretch injury in the cell lysates of hBMVEC treated with apocynin (NADPH oxidase inhibitor), VEGF-A (recombinant human VEGF), Ki8751 (inhibitor of VEGFR phosphorylation), and TIMP1 (MMPs inhibitor). Bar graph represents the quantification of ICAM-1 versus ß-actin (n = 6/group). I–K, Western blot analysis of NOX1, 4HNE, MMP-2, MMP-9, and ß-actin 24 h after 3.0 psi stretch injury in the cell lysates of hBMVEC treated with apocynin, VEGF-A, Ki8751, and TIMP1. Bar graph represents the quantification of ICAM-1 versus ß-actin (n = 6/group). L–N, Western blot analysis of VEGF-A, VEGFR-2, p-VEGFR-2Y1059, p-VEGFR-2Y1175, and ß-actin 24 h after 3.0 psi stretch injury in the cell lysates of hBMVEC treated with apocynin, VEGF-A, Ki8751, and TIMP1 (n = 6/group). All values are expressed as mean ± SD. Statistically significant, ***p < 0.001 versus uninjured group; ##p < 0.01, ###p < 0.001 versus injury group.

Fig 5: TBI activates ICAM-1 protein in both mild and moderate injury. A, Representative illustration of injury site and tissue sampling area in the mice brain subjected to FPI. The injury site, PFC, and hippocampus are labeled with red, blue, and green colors, respectively. B, Immunofluorescent staining of ICAM-1 (red) in the hippocampus area and merged with GLUT-1 (green) and DAPI (blue) after 10 and 20 psi FPI. Scale bar: 100 µm (shown in c is for a–c, first column) and 40 µm (shows in c3 is for a1–a3, b1–b3, and c1–c3, columns 2–4). C, Quantification of ICAM-1 staining in the hippocampus area of WT uninjured, 10 and 20 psi FPI mice using ImageJ software (n = 4/group). D, E, Western blot analysis of ICAM-1 and ß-actin in the tissue lysates from PFC and hippocampus of WT and ICAM-1-/- mice 48 h after 10 and 20 psi FPI. The bar graph with dot plots shows the quantification of ICAM-1 versus ß-actin (n = 6/group). F–H, ELISA quantification of ICAM-1 in PFC (F), hippocampus (G) tissue lysates and blood plasma (H) 48 h and 14 d following 10 and 20 psi FPI (n = 6/group). I, J, mRNA expression level of ICAM-1 using qPCR from PFC and hippocampus of WT and ICAM-1-/- mice 48 h and 14 d after 10 and 20 psi FPI (n = 6/group). K, L, Western blot analysis of ICAM-1 and ß-actin expression at different time points (6 h, 12 h, 24 h, 48 h, and 14 d) in the PFC (K) and hippocampus (L) of WT uninjured and 20 psi FPI. Bar graph represents the densitometric ratio of ICAM-1 bands versus ß-actin bands (n = 6/group). M, ELISA quantification of ICAM-1 at different time points (6 h, 12 h, 24 h, 48 h, and 14 d) in the blood plasma of WT uninjured and 20 psi FPI mice (n = 6/group). All values are expressed as mean ± SD. Statistically significant *p < 0.05, **p < 0.01, ***p < 0.001 versus WT uninjured group; #p < 0.05, ###p < 0.001 between 10 and 20 psi; $p < 0.05, $$p < 0.01 between time points; ns = non-significant.