Published online before print January 31, 2002, doi: 10.1161/01.ATV.0000012262.76205.6A
© 2002 American Heart Association, Inc.
Vascular Biology |
Role of Mitochondrial Oxidant Generation in Endothelial Cell Responses to Hypoxia
From the Departments of Medicine and Surgery, The University of Chicago, Chicago, Ill.
Correspondence to Paul T. Schumacker, PhD, Department of Medicine, MC6026, 5841 S Maryland Ave, Chicago, IL 60637. E-mail pschumac{at}medicine.bsd.uchicago.edu
Endothelial cells increase their secretion of the cytokine interleukin-6 (IL-6) during hypoxia, which then acts in an autocrine fashion to increase the permeability of cell monolayers. These responses are attenuated by antioxidants, suggesting that reactive oxygen species (ROS) participate in signaling in hypoxic endothelium. We tested whether mitochondria are responsible for these ROS in human umbilical vein endothelial cells exposed to hypoxia. Oxidation of the probe 2', 7'-dichlorodihydrofluorescein to fluorescent dichlorofluorescein or the probe dihydroethidium was used to assess oxidant signaling, whereas permeability was assessed by using transendothelial electrical resistance. Hypoxia elicited increases in dichlorofluorescein and dihydroethidium fluorescence that were abrogated by the mitochondrial electron transport (ET) inhibitors rotenone (2 µmol/L) and diphenyleneiodonium (5 µmol/L). The same ET inhibitors also attenuated hypoxia-induced increases in nuclear factor-
B (NF-B) activation, although they did not abrogate NF-B activation in response to endotoxin (lipopolysaccharide). ET inhibition also abolished the hypoxia-induced increases in IL-6 mRNA expression, hypoxia-stimulated IL-6 secretion into the media, and the hypoxia-induced increases in transendothelial electrical resistance of human umbilical vein endothelial cell monolayers. By contrast, the above responses to hypoxia were not significantly affected by treatment with the NAD(P)H oxidase inhibitor apocynin (30 µmol/L), the xanthine oxidase inhibitor allopurinol (100 µmol/L), or the NO synthase inhibitor N-nitro-L-arginine (100 µmol/L). We conclude that ROS signals originating from the mitochondrial ET chain trigger the increase in NF-B activation, the transcriptional activation of IL-6, the secretion of IL-6 into the cell culture media, and the increases in endothelial permeability observed during hypoxia.
Key Words: reactive oxygen species • human umbilical vein endothelial cells • ischemia • signal transduction • microcirculation
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