IgG dose-dependently decreased the intracellular superoxide (O2·−) level in human aortic endothelial cells (HAECs). A, Photomicrographs of intracellular O2·− in HAECs (dihydroethidium [DHE] staining, red fluorescence) taken using confocal microscopy. HAECs were treated with IgG (10 mg/mL) for 24 hours. Untreated cells were used as the control. B, The intracellular O2·− level (mean intensity of DHE staining by flow cytometry) after treatments with various doses of IgG. For the dose-response study, HAECs were incubated with different doses (0.5, 5.0, 10.0, and 15.0 mg/mL) of normal human IgG for 48 hours. The cells were washed twice in PBS and then incubated with DHE/PBS at 37°C for 15 minutes before analysis of the O2·− level using flow cytometry (10000 cells gated). The O2·− level was expressed as mean intensity (MI) of DHE staining. C, The time course of changes in the O2·− level after treatment with IgG (10 mg/mL). The most effective dose (10 mg/mL) of IgG was chosen for the time course study. The cells were incubated with 10 mg/mL of IgG for 24, 48, and 72 hours. Original magnification, ×630. The data were from ≥3 independent experiments (n=3). *P<0.05; **P<0.01; ***P<0.001.
IgG upregulated AMP-activated protein kinase (AMPK) activity but downregulated NADPH oxidase activity. A, Western blot analysis of phosphorylated (p-)AMPKα1. B, Western blot analysis of AMPKα1 expression. C, NADPH oxidase activity (arrows indicating addition of NADPH). RLU indicates relative light unit. D, Intracellular NO measured by 4,5-Diaminofluorescein diacetate (DAF-2 DA) fluorescence using flow cytometry. n=3 independent experiments. *P<0.05; **P<0.01; ***P<0.001.
IgG decreased the intracellular superoxide (O2·−) level via AMP-activated protein kinase (AMPK). A, Western blot analysis of phosphorylated (p)-AMPK. B, Intracellular O2·− (dihydroethidium [DHE] staining) measured by flow cytometry. n=3 independent experiments. *P<0.05; **P<0.01; ***P<0.001. AI indicates AMPK inhibitor.
IgG downregulated phosphorylated (p)-Rac1 and NADPH oxidase activities via AMP-activated protein kinase (AMPK). A, Western blot analysis of p-Rac1. B, Western blot analysis of Rac1. C, NADPH oxidase activity (arrows indicating addition of NADPH). RLU indicates relative light unit. n=3 independent experiments. **P<0.01, ***P<0.001 vs the untreated group.
IgG upregulated superoxide dismutase (SOD) protein expression via AMP-activated protein kinase (AMPK). A, Western blot analysis of MnSOD protein expression. B, Western blot analysis of endothelial NO synthase (eNOS) protein expression. n=3 independent experiments. **P<0.01. AI indicates AMPK inhibitor.
IgG attenuated human aortic endothelial cells (HAEC) migration via AMP-activated protein kinase (AMPK). HAECs were treated with IgG for 24 hours. The bio-gels were then removed and allowed the cells to migrate for 20 hours. A, Photomicrographs of HAEC migration were taken before removal of bio-gel (0 hour) and at 2, 5, 10, and 20 hours after removal of bio-gel, respectively. B, Percentage of migration at 2 hours. C, Percentage of migration at 5 hours. D, Percentage of migration at 10 hours. E, Percentage of migration at 20 hours. n=3 independent experiments. *P<0.05; **P<0.01. AI indicates AMPK inhibitor. Phase contrast micrographs, ×100.
IgG decreased cell permeability and stress fiber formation via AMP-activated protein kinase (AMPK). A, The time course of fluorescein isothiocyanate (FITC)-dextran leakage (arrow shows obvious change of FITC-dextran leakage at 15-minute time point; RFUs indicates relative fluorescence units). B, Cell permeability assessed by percentage change in FITC-dextran leakage at the 15-minute time point. C, Representative photomicrographs of stress fiber formation (F-actin, green fluorescence). D, Quantification of F-actin formation. n=3 independent experiments. *P<0.05, **P<0.01. AI indicates AMPK inhibitor. Magnification, ×630. Scale bar, 50 μm.
IgG upregulated vascular endothelial (VE) cadherin expression not through AMP-activated protein kinase (AMPK). A, Western blot analysis of VE cadherin protein expression. B, In situ expression of VE cadherin (red, arrows) by immunocytochemistry and immunofluorescence. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). VE cadherin was mainly localized in the cell membrane (single arrow) and the cell-cell junction area (doublearrows). C, Quantification of VE cadherin expression based on VE-cadherin fluorescence intensity. VE-C indicates VE cadherin. Magnification, ×630. Scale bar, 50 μm.
IgG dose-dependently decreased the intracellular superoxide (O2·−) level in human aortic endothelial cells (HAECs). A, Photomicrographs of intracellular O2·− in HAECs (dihydroethidium [DHE] staining, red fluorescence) taken using confocal microscopy. HAECs were treated with IgG (10 mg/mL) for 24 hours. Untreated cells were used as the control. B, The intracellular O2·− level (mean intensity of DHE staining by flow cytometry) after treatments with various doses of IgG. For the dose-response study, HAECs were incubated with different doses (0.5, 5.0, 10.0, and 15.0 mg/mL) of normal human IgG for 48 hours. The cells were washed twice in PBS and then incubated with DHE/PBS at 37°C for 15 minutes before analysis of the O2·− level using flow cytometry (10000 cells gated). The O2·− level was expressed as mean intensity (MI) of DHE staining. C, The time course of changes in the O2·− level after treatment with IgG (10 mg/mL). The most effective dose (10 mg/mL) of IgG was chosen for the time course study. The cells were incubated with 10 mg/mL of IgG for 24, 48, and 72 hours. Original magnification, ×630. The data were from ≥3 independent experiments (n=3). *P<0.05; **P<0.01; ***P<0.001.
IgG upregulated AMP-activated protein kinase (AMPK) activity but downregulated NADPH oxidase activity. A, Western blot analysis of phosphorylated (p-)AMPKα1. B, Western blot analysis of AMPKα1 expression. C, NADPH oxidase activity (arrows indicating addition of NADPH). RLU indicates relative light unit. D, Intracellular NO measured by 4,5-Diaminofluorescein diacetate (DAF-2 DA) fluorescence using flow cytometry. n=3 independent experiments. *P<0.05; **P<0.01; ***P<0.001.
