Fig 1: Mevastatin (MVS)-induced heme oxygenase (HO)-1 expression attenuates tumor necrosis factor α (TNFα)-stimulated phosphorylation of NF-κB p65 and VCAM-1 expression. (A) Human pulmonary alveolar epithelial cells (HPAEpiCs)s were incubated with different concentrations of TNFα (15, 5, 1, or 0.3 ng/mL) for the indicated time intervals. (B,C) Cells were pretreated different concentrations of Bay11-7082 (0.1, 1, or 10 μM) for 1 h, and then incubated with or without TNFα (15 ng/mL) for 24 h (protein) or 4 h (mRNA). (D) HPAEpiCs were pretreated with 30 μM MVS for 1 h, incubated with or without tin protoporphyrin IX (SnPPIX) (0.1 μM) for 1 h, and then stimulated with TNFα for 24 h. (E) Cells were pretreated with 30 μM MVS for 24 h and then incubated with TNFα (15 ng/mL) for the indicated time intervals. (A,B,D,E) The levels of VCAM-1, β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phospho-p65, p65, and HO-1 protein were determined by Western blot using respective antibodies as indicated. Data are expressed as mean ± SEM of five independent experiments (n = 5). # p < 0.01, as compared with the cells exposed to the indicated reagents.
Fig 2: A schematic pathway for MVS-induced HO-1 expression protecting against the inflammatory responses in HPAEpiCs challenged with TNFα. MVS-induced HO-1 expression was mediated via PKCα/Pyk2/p38 MAPK or JNK1/2-dependent phosphorylation of c-Jun binding to AP-1 binding sites on HO-1 promoter that attenuated the TNFα-stimulated VCAM-1 expression and activation of NF-κB p65.
Fig 3: TGF-β1-stimulated upregulation of VCAM-1 is dependent on Smad pathway. The silence efficiency of si-Smad3 was confirmed by western blot assay (a). Endometriotic cyst stromal cells were transfected with si-Smad3, and then treated with recombinant human TGF-β1 (10 ng/mL) for 48 h. Cells were harvested for western blot assay (b). **P < 0.01 and ***P < 0.001
Fig 4: Effects of E2 on the levels of adherence and angiogenic factors. (A) Representative western blot images demonstrating the expression of CD49d, ICAM1 and LFA-1 in the four groups. (B) Relative protein expression levels of CD49d, ICAM1 and LFA-1. (C) Representative western blot images demonstrating the expression of MMP2, MMP9 and VCAM1 in the placenta in the four groups. (D) Relative protein expression of MMP2, MMP9 and VCAM1. n=12 rats/group. *P<0.05, **P<0.01 and ***P<0.001 vs. control; ##P<0.01 and ###P<0.001 vs. L-NAME and L-NAME + NS. E2, estradiol; ICAM1, intercellular adhesion molecule-1; LFA-1, leukocyte function-associated antigen; MMP, matrix metallopeptidase; VCAM1, vascular cell adhesion molecule-1; L-NAME, N (omega)-nitro-L-arginine methyl ester; NS, normal saline.
Fig 5: ESMIRO/ApoE−/−/Nox2−/y mice have increased lipid deposition in the thoraco-abdominal aorta and multiple foci of elastin fragmentation. A: greater lipid deposition in thoraco-abdominal aorta of ESMIRO/ApoE−/−/Nox2−/y (n = 21) compared with ESMIRO/ApoE−/−/Nox+/y mice (n = 15). Scale bar = 250 µm. B: no difference in atherosclerosis at the level of the aortic sinus of ESMIRO/ApoE−/−/Nox2−/y (n = 7) compared with ESMIRO/ApoE−/−/Nox+/y mice (n = 6). Scale bar = 500 µm. C: increased elastin fragmentation in aortic wall at the level of the aortic sinus in ESMIRO/ApoE−/−/Nox2−/y (n = 8) compared with ESMIRO/ApoE−/−/Nox+/y mice (n = 6). Scale bar = 500 µm. D: similar size populations of circulating leukocytes in ESMIRO/ApoE−/−/Nox2−/y (n = 13) compared with ESMIRO/ApoE−/−/Nox2+/y mice (n = 11). E: measurement of Nox4 NADPH oxidase, superoxide dismutase 2 (SOD2) and catalase mRNA level showed no difference between ESMIRO/ApoE−/−/Nox2−/y (n = 10) mice and ESMIRO/ApoE−/−/Nox2+/y mice (n = 9). F: measurement of mRNA level showed no difference in interleukin 1β (IL-1β), tumor necrosis factor-α (TNF-α), the chemokines CCL2 and CCR2, adhesion molecules intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion protein 1 (VCAM-1) expression in ESMIRO/ApoE−/−/Nox2−/y (n = 8–10) compared with ESMIRO/ApoE−/−/Nox+/y mice (n = 8–9). ESMIRO, endothelium-specific mutant insulin receptor-overexpressing mice; ApoE, apolipoprotein E; Nox2, Nox2 isoform of NADPH oxidase; AU, arbitrary units. Data are expressed as mean ± SE; n = number of mice per genotype. *P < 0.05.
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