title: Chronic hypoxia promotes pulmonary artery endothelial cell proliferation through H2O2-induced 5-lipoxygenase.
abstract: Pulmonary Hypertension (PH) is a progressive disorder characterized by endothelial dysfunction and proliferation. Hypoxia induces PH by increasing vascular remodeling. A potential mediator in hypoxia-induced PH development is arachidonate 5-Lipoxygenase (ALOX5). While ALOX5 metabolites have been shown to promote pulmonary vasoconstriction and endothelial cell proliferation, the contribution of ALOX5 to hypoxia-induced proliferation remains unknown. We hypothesize that hypoxia exposure stimulates HPAEC proliferation by increasing ALOX5 expression and activity. To test this, human pulmonary artery endothelial cells (HPAEC) were cultured under normoxic (21% O2) or hypoxic (1% O2) conditions for 24-, 48-, or 72 hours. In a subset of cells, the ALOX5 inhibitor, zileuton, or the 5-lipoxygenase activating protein inhibitor, MK-886, was administered during hypoxia exposure. ALOX5 expression was measured by qRT-PCR and western blot and HPAEC proliferation was assessed. Our results demonstrate that 24 and 48 hours of hypoxia exposure have no effect on HPAEC proliferation or ALOX5 expression. Seventy two hours of hypoxia significantly increases HPAEC ALOX5 expression, hydrogen peroxide (H2O2) release, and HPAEC proliferation. We also demonstrate that targeted ALOX5 gene silencing or inhibition of the ALOX5 pathway by pharmacological blockade attenuates hypoxia-induced HPAEC proliferation. Furthermore, our findings indicate that hypoxia-induced increases in cell proliferation and ALOX5 expression are dependent on H2O2 production, as administration of the antioxidant PEG-catalase blocks these effects and addition of H2O2 to HPAEC promotes proliferation. Overall, these studies indicate that hypoxia exposure induces HPAEC proliferation by activating the ALOX5 pathway via the generation of H2O2.
authors.affiliation: Emory University School of Medicine/Atlanta Veterans Affairs Medical Center, Department of Pulmonary, Allergy and Critical Care Medicine, Atlanta, Georgia, United States of America.
authors.forename: Kristi M
authors.initials: KM
authors.lastname: Porter
authors.affiliation: Emory University School of Medicine/Atlanta Veterans Affairs Medical Center, Department of Pulmonary, Allergy and Critical Care Medicine, Atlanta, Georgia, United States of America.
authors.forename: Bum-Yong
authors.initials: BY
authors.lastname: Kang
authors.affiliation: Emory University School of Medicine/Atlanta Veterans Affairs Medical Center, Department of Pulmonary, Allergy and Critical Care Medicine, Atlanta, Georgia, United States of America.
authors.forename: Sherry E
authors.initials: SE
authors.lastname: Adesina
authors.affiliation: Emory University School of Medicine/Atlanta Veterans Affairs Medical Center, Department of Pulmonary, Allergy and Critical Care Medicine, Atlanta, Georgia, United States of America.
authors.forename: Tamara C
authors.initials: TC
authors.lastname: Murphy
authors.affiliation: Emory University School of Medicine/Atlanta Veterans Affairs Medical Center, Department of Pulmonary, Allergy and Critical Care Medicine, Atlanta, Georgia, United States of America.
authors.forename: C Michael
authors.initials: CM
authors.lastname: Hart
authors.affiliation: Emory University School of Medicine/Atlanta Veterans Affairs Medical Center, Department of Pulmonary, Allergy and Critical Care Medicine, Atlanta, Georgia, United States of America.
authors.forename: Roy L
authors.initials: RL
authors.lastname: Sutliff
mesh_terms: D001094:Arachidonate 5-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D015687:Cell Hypoxia; D002460:Cell Line; D049109:Cell Proliferation; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006918:Hydroxyurea / Q000031:analogs & derivatives / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism*; D011651:Pulmonary Artery / Q000166:cytology; D002722:Chlorobenzenes
chemical_list: D001094:Arachidonate 5-Lipoxygenase; D006861:Hydrogen Peroxide; D006918:Hydroxyurea; D007211:Indoles; D016859:Lipoxygenase Inhibitors; D010100:Oxygen; C060893:MK-886; D002722:Chlorobenzenes; C583212:ALOX5 protein, human; C063449:zileuton