Expression of NADPH oxidases and enhanced H(2)O(2)-generating activity in human coronary artery endothelial cells upon induction with tumor necrosis factor-alpha.
Tumor necrosis factor (TNF)-alpha, which potentiates reactive oxygen species (ROS) generation, is crucial for the development of coronary arteritis and aneurysm in Kawasaki disease. We hypothesized that vascular NADPH oxidase (Nox) enzymes participate in the TNF-alpha-triggered endothelial damage through elevating ROS generation. Thus, we herein examine the expression of Nox enzymes in human coronary artery endothelial cells (HCAEC) and the effects of TNF-alpha on Nox-mediated ROS generation. We show that HCAEC in culture spontaneously generate H(2)O(2) at basal level (0.53 nmol/min/mg protein). In searching for Nox components responsible for the H(2)O(2) generation, two distinct isoforms of Nox4 are found expressed in HCAEC: the prototype Nox4A and the shorter Nox4B, respectively in the postnuclear supernatant and the nuclear fractions. Other expressed Nox family components are: as mRNAs, Nox4C, Nox4D, Nox1, p51(nox), and Racs; as mRNAs and proteins, Nox2, p22(phox), p47(phox), and p67(phox). The H(2)O(2)-generating activity increases up to three-fold upon inclusion of TNF-alpha in culture, concomitantly with augmented expressions of Nox4A, p22(phox), p47(phox) and p67(phox) proteins. Together, these results suggest that Nox2 and Nox4A enzymes are induced by TNF-alpha endowing HCAEC with enhanced ROS-generating activity, which may play a role in the initial endothelial dysfunction through oxidative stress.
Yoshida
Lucia S
LS
Department of Infectious Diseases, National Research Institute for Child Health and Development, Tokyo, Japan.
Tsunawaki
Shohko
S
D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology*; D004730:Endothelium, Vascular / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D006861:Hydrogen Peroxide; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human