title: Role of mitochondrial oxidant generation in endothelial cell responses to hypoxia.
abstract: 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 micromol/L) and diphenyleneiodonium (5 micromol/L). The same ET inhibitors also attenuated hypoxia-induced increases in nuclear factor-kappaB (NF-kappaB) activation, although they did not abrogate NF-kappaB 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 micromol/L), the xanthine oxidase inhibitor allopurinol (100 micromol/L), or the NO synthase inhibitor N-nitro-L-arginine (100 micromol/L). We conclude that ROS signals originating from the mitochondrial ET chain trigger the increase in NF-kappaB 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.
authors.affiliation: Departments of Medicine and Surgery, The University of Chicago, Chicago, Illinois, USA.
authors.forename: Daryl P
authors.initials: DP
authors.lastname: Pearlstein
authors.forename: Mir H
authors.initials: MH
authors.lastname: Ali
authors.forename: Paul T
authors.initials: PT
authors.lastname: Mungai
authors.forename: Karen L
authors.initials: KL
authors.lastname: Hynes
authors.forename: Bruce L
authors.initials: BL
authors.lastname: Gewertz
authors.forename: Paul T
authors.initials: PT
authors.lastname: Schumacker
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D000493:Allopurinol / Q000494:pharmacology; D000704:Analysis of Variance; D000975:Antioxidants / Q000494:pharmacology; D002199:Capillary Permeability; D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004996:Ethidium / Q000031:analogs & derivatives* / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology; D010084:Oxidation-Reduction; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012402:Rotenone / Q000494:pharmacology; D014471:Umbilical Veins
chemical_list: C110400:2',7'-dichlorodihydrofluorescein diacetate; D000098:Acetophenones; D000975:Antioxidants; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015850:Interleukin-6; D016328:NF-kappa B; D009861:Onium Compounds; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D012402:Rotenone; C067883:dihydroethidium; D000493:Allopurinol; C007517:diphenyleneiodonium; C056165:acetovanillone; D004996:Ethidium