CHEMILUMINESCENCE STUDY ON THE REGULATION OF NADPH OXIDASE ACTIVITY BY THIOREDOXIN REDUCTASE IN VASCULAR ENDOTHELIAL CELLS

Low-level arsenite treatment of porcine aortic endothelial cells (PAEC) stimulated superoxide accumulation that was attenuated by inhibitors of NAD(P)H oxidase. To demonstrate whether arsenite stimulated NADPH oxidase, intact PAEC were treated with arsenite for up to 2 h and membrane fractions were prepared to measure NADPH oxidase activity. Arsenite (5 μM) stimulated a twofold increase in activity by 1 h, which was inhibited by the oxidase inhibitor diphenyleneiodonium chloride. Direct treatment of isolated membranes with arsenite had no effect. Analysis of NADPH oxidase components revealed that p67phoxlocalized exclusively to membranes of both control and treated cells. In contrast, cytosolic Rac1 translocated to the membrane fractions of cells treated with arsenite or angiotensin II but not with tumor necrosis factor. Immunodepletion of p67phoxblocked oxidase activity stimulated by all three compounds. However, depleting Rac1 inhibited responses only to arsenite and angiotensin II. These data demonstrate that stimulus-specific activation of NADPH oxidase in endothelial cells was the source of reactive oxygen in endothelial cells after noncytotoxic arsenite exposure.

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Cigarette smoke extract induces HO-1 expression in mouse cerebral vascular endothelial cells: Involvement of c-Src/NADPH oxidase/PDGFR/JAK2/STAT3 pathway

Journal of Cellular Physiology ◽

10.1002/jcp.22270 ◽

2010 ◽

Vol 225 (3) ◽

pp. 741-750 ◽

Cited By ~ 23

Author(s):

Ruey-Horng Shih ◽

I-Ta Lee ◽

Hsi-Lung Hsieh ◽

Yu Ru Kou ◽

Chuen-Mao Yang

Keyword(s):

Endothelial Cells ◽

Nadph Oxidase ◽

Cigarette Smoke ◽

Vascular Endothelial Cells ◽

Cigarette Smoke Extract ◽

Vascular Endothelial ◽

Stat3 Pathway ◽

Cerebral Vascular

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Abstract 776: Protein Disulfide Isomerase Is Involved In Redox Regulation Of Nitric Oxide Output During Laminar Shear Stress In Endothelial Cells

Circulation ◽

10.1161/circ.116.suppl_16.ii_149-b ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Denise C Fernandes ◽

Celio X Santos ◽

Hanjoong Jo ◽

Francisco R Laurindo

Keyword(s):

Endothelial Cells ◽

Nadph Oxidase ◽

Oxidase Activity ◽

Membrane Fraction ◽

Protein Disulfide Isomerase ◽

Disulfide Isomerase ◽

Nadph Oxidase Activity ◽

Caveolin 1 ◽

Protein Disulfide ◽

Laminar Shear

While anti-atherogenic effects of sustained laminar shear (LS) involve NO release from eNOS, increases in LS trigger transient superoxide production via NADPH oxidase. Recently, we showed that NADPH oxidase undergoes thiol-dependent regulation by the thioredoxin superfamily chaperone Protein Disulfide Isomerase (PDI). PDI is known to promote NO internalization via trans-nitrosation reactions. We hypothesized that PDI-dependent support of NADPH oxidase activity affects NO output during sustained LS. Cultured rabbit aortic endothelial cells (RAEC) submitted to LS (15 dynes/cm 2 ) in a cone-plate system for 18h exhibited (vs. static controls): Decreased (~50%) superoxide production (HPLC analysis of DHE oxidation); Decreased (~20%) NADPH-triggered hydrogen peroxide production in membrane fraction (Amplex Red assay); Decreased mRNA expression of Nox1 (67%) and Nox4 (45%) (real-time QPCR); Increased eNOS expression (~50%, western blot) and nitrite levels in culture medium (Δ = 7.1±2.5[SD] μM, NO Analyzer and Griess reaction); Decrease in total and membrane fraction PDI protein expression (~20%) without changes in membrane fraction/total ratio of PDI. RAEC were transfected with c-myc -tagged plasmid coding for wild-type (WT) PDI or PDI mutated in 4 thioredoxin-motif cysteine residues. Forced expression (2-fold) of mutated but not WT PDI led to increase in nitrite output after LS (18h) (Δmutated = 17.2±3.3 μM vs. ΔWT = 7.0±1.9 μM, n=3, p<0.02). Confocal microscopy indicated similar subcellular localization between WT and mutated PDI. PDI co-imunoprecipitated with p22phox NADPH oxidase subunit, but not with eNOS or caveolin-1, either in static condition or after LS. Fractionation studies in sucrose gradients showed that PDI is distributed throughout several fractions in static conditions, including caveolin-1-enriched fractions, but migrates to higher-density fractions, not containing caveolin-1, during sustained LS. These results suggest that PDI is involved in regulation of NO output during LS via its effects on NADPH oxidase activity.

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Protective Role of Endogenous Kallistatin in Vascular Injury and Senescence by Inhibiting Oxidative Stress and Inflammation

Oxidative Medicine and Cellular Longevity ◽

10.1155/2018/4138560 ◽

2018 ◽

Vol 2018 ◽

pp. 1-8 ◽

Cited By ~ 10

Author(s):

Julie Chao ◽

Youming Guo ◽

Lee Chao

Keyword(s):

Oxidative Stress ◽

Endothelial Cells ◽

Nadph Oxidase ◽

Vascular Injury ◽

Oxidase Activity ◽

Sirtuin 1 ◽

Mouse Lung ◽

Heparin Binding ◽

Diabetic Mice ◽

Nadph Oxidase Activity

Kallistatin was identified in human plasma as a tissue kallikrein-binding protein and a serine proteinase inhibitor. Kallistatin exerts pleiotropic effects on angiogenesis, oxidative stress, inflammation, apoptosis, fibrosis, and tumor growth. Kallistatin levels are markedly reduced in patients with coronary artery disease, sepsis, diabetic retinopathy, inflammatory bowel disease, pneumonia, and cancer. Moreover, plasma kallistatin levels are positively associated with leukocyte telomere length in young African Americans, indicating the involvement of kallistatin in aging. In addition, kallistatin treatment promotes vascular repair by increasing the migration and function of endothelial progenitor cells (EPCs). Kallistatin via its heparin-binding site antagonizes TNF-α-induced senescence and superoxide formation, while kallistatin’s active site is essential for inhibiting miR-34a synthesis, thus elevating sirtuin 1 (SIRT1)/eNOS synthesis in EPCs. Kallistatin inhibits oxidative stress-induced cellular senescence by upregulating Let-7g synthesis, leading to modulate Let-7g-mediated miR-34a-SIRT1-eNOS signaling pathway in human endothelial cells. Exogenous kallistatin administration attenuates vascular injury and senescence in association with increased SIRT1 and eNOS levels and reduced miR-34a synthesis and NADPH oxidase activity, as well as TNF-α and ICAM-1 expression in the aortas of streptozotocin- (STZ-) induced diabetic mice. Conversely, endothelial-specific depletion of kallistatin aggravates vascular senescence, oxidative stress, and inflammation, with further reduction of Let-7g, SIRT1, and eNOS and elevation of miR-34a in mouse lung endothelial cells. Furthermore, systemic depletion of kallistatin exacerbates aortic injury, senescence, NADPH oxidase activity, and inflammatory gene expression in STZ-induced diabetic mice. These findings indicate that endogenous kallistatin displays a novel role in protection against vascular injury and senescence by inhibiting oxidative stress and inflammation.

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