title: Glutathione adducts induced by ischemia and deletion of glutaredoxin-1 stabilize HIF-1α and improve limb revascularization.
abstract: Reactive oxygen species (ROS) are increased in ischemic tissues and necessary for revascularization; however, the mechanism remains unclear. Exposure of cysteine residues to ROS in the presence of glutathione (GSH) generates GSH-protein adducts that are specifically reversed by the cytosolic thioltransferase, glutaredoxin-1 (Glrx). Here, we show that a key angiogenic transcriptional factor hypoxia-inducible factor (HIF)-1α is stabilized by GSH adducts, and the genetic deletion of Glrx improves ischemic revascularization. In mouse muscle C2C12 cells, HIF-1α protein levels are increased by increasing GSH adducts with cell-permeable oxidized GSH (GSSG-ethyl ester) or 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanyl thiocarbonylamino) phenylthiocarbamoylsulfanyl] propionic acid (2-AAPA), an inhibitor of glutathione reductase. A biotin switch assay shows that GSSG-ester-induced HIF-1α contains reversibly modified thiols, and MS confirms GSH adducts on Cys(520) (mouse Cys(533)). In addition, an HIF-1α Cys(520) serine mutant is resistant to 2-AAPA-induced HIF-1α stabilization. Furthermore, Glrx overexpression prevents HIF-1α stabilization, whereas Glrx ablation by siRNA increases HIF-1α protein and expression of downstream angiogenic genes. Blood flow recovery after femoral artery ligation is significantly improved in Glrx KO mice, associated with increased levels of GSH-protein adducts, capillary density, vascular endothelial growth factor (VEGF)-A, and HIF-1α in the ischemic muscles. Therefore, Glrx ablation stabilizes HIF-1α by increasing GSH adducts on Cys(520) promoting in vivo HIF-1α stabilization, VEGF-A production, and revascularization in the ischemic muscles.
authors.affiliation: Vascular Biology Section, Boston University School of Medicine, Boston, MA 20118; Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA 20118;
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Watanabe
authors.affiliation: Vascular Biology Section, Boston University School of Medicine, Boston, MA 20118; Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA 20118;
authors.forename: Colin E
authors.initials: CE
authors.lastname: Murdoch
authors.affiliation: Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA 20118;
authors.forename: Soichi
authors.initials: S
authors.lastname: Sano
authors.affiliation: Section of Endocrinology, Boston University School of Medicine, Boston, MA 20118.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ido
authors.affiliation: Vascular Biology Section, Boston University School of Medicine, Boston, MA 20118; Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA 20118;
authors.forename: Markus M
authors.initials: MM
authors.lastname: Bachschmid
authors.affiliation: Vascular Biology Section, Boston University School of Medicine, Boston, MA 20118; Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA 20118;
authors.forename: Richard A
authors.initials: RA
authors.lastname: Cohen
authors.affiliation: Vascular Biology Section, Boston University School of Medicine, Boston, MA 20118; Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA 20118; rmatsui@bu.edu.
authors.forename: Reiko
authors.initials: R
authors.lastname: Matsui
mesh_terms: D000818:Animals; D015687:Cell Hypoxia; D054477:Glutaredoxins / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D057809:HEK293 Cells; D006614:Hindlimb / Q000098:blood supply* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D007511:Ischemia / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D018345:Mice, Knockout; D018482:Muscle, Skeletal / Q000098:blood supply / Q000378:metabolism / Q000473:pathology; D055550:Protein Stability; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics
chemical_list: C516005:GLRX protein, human; C516006:Glrx protein, mouse; D054477:Glutaredoxins; C497442:HIF1A protein, human; C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D005978:Glutathione
keywords: GSH-protein adducts; S-glutathionylation; glutaredoxin-1; hypoxia-inducible factor-1; ischemic limb revascularization