Figure 2. 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 β-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 β-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 β-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 β-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 β-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 β-actin (n = 6/group). I–K, Western blot analysis of NOX1, 4HNE, MMP-2, MMP-9, and β-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 β-actin (n = 6/group). L–N, Western blot analysis of VEGF-A, VEGFR-2, p-VEGFR-2Y1059, p-VEGFR-2Y1175, and β-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.