Effect of NADPH oxidase inhibition on E-selectin expression induced by concomitant anoxia/reoxygenation and TNF-alpha.
The aim of this study was to quantify the expression of E-selectin, intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vascular endothelial cells (HUVECs) exposed to anoxia/reoxygenation (A/R) in the presence or absence of an inflammatory context (0.1 IU/ml tumor necrosis factor-alpha [TNF-alpha]) and to investigate the effects of two different NADPH inhibitors, apocynin and diphenyleneiodonium (DPI), on the expression of the endothelial cell adhesion molecules. Confluent HUVECs were exposed to anoxia for 3 hours (100% N2), followed by a reoxygenation period of 4 hours. TNF-alpha at 0.1 IU/ml was added to the medium either under normoxic conditions for 7 hours (TNF-alpha) or just before the start of anoxia (A/R + TNF-alpha). Levels of E-selectin, VCAM-1, and ICAM-1 were quantified using specific monoclonal antibodies revealed by an alkaline phosphatase-labeled goat F(ab)'2 fragment against mouse IgG antibody and the fluorescent substrate Attophos. Adhesion experiments were also performed using calcein-labeled U937 leukocytes. HUVECs submitted to A/R overexpressed E-selectin but not VCAM-1 or ICAM-1, whereas TNF-alpha at 0.1 IU/ ml increased the expression of all three adhesion molecules. In endothelial cells subjected to A/R in the presence of TNF-alpha, a synergistic increase of E-selectin expression and a synergistic adhesion of U937 cells was noted. The NADPH oxidase inhibitors apocynin and DPI both decreased significantly the U937 adhesion and the E-selectin overexpression on HUVECs submitted to A/R, TNF-alpha, or A/R + TNF-alpha. These results suggest that E-selectin expression is implicated in the leukocyte adhesion to HUVECs caused by A/R in the presence or absence of an inflammatory context. NADPH oxidase appears to participate in the E-selectin overexpression on HUVECs subjected either to A/R and/or TNF-alpha, suggesting a major role of this enzyme in the ischemia/reperfusion syndrome.
Paysant
Jérôme R
JR
Division of Angiology, Servier Research Institute, Suresnes, France.
Rupin
Alain
A
Verbeuren
Tony J
TJ
D001161:Arteriosclerosis / Q000201:enzymology / Q000503:physiopathology; D002448:Cell Adhesion / Q000502:physiology*; D002634:Chemotaxis, Leukocyte / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions / Q000502:physiology; D019040:E-Selectin / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000201:enzymology* / Q000503:physiopathology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D000860:Hypoxia / Q000201:enzymology* / Q000503:physiopathology; D007150:Immunohistochemistry; D007249:Inflammation / Q000201:enzymology* / Q000503:physiopathology; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015427:Reperfusion Injury / Q000201:enzymology / Q000503:physiopathology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology; D020298:U937 Cells; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
D019040:E-Selectin; D004791:Enzyme Inhibitors; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1; D019255:NADPH Oxidases