Journal Article

Glutathione adducts induced by ischemia and deletion of glutaredoxin-1 stabilize HIF-1α and improve limb revascularization

Watanabe, Yosuke, Bachschmid, Markus M., Matsui, Reiko, Murdoch, Colin E., Ido, Yasuo, Cohen, Richard A., Sano, Soichi

2016

Overview

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 Cys520 (mouse Cys533). In addition, an HIF-1α Cys520 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 Cys520 promoting in vivo HIF-1α stabilization, VEGF-A production, and revascularization in the ischemic muscles.

• Publisher: National Academy of Sciences
• Subject: Animals / Biological Sciences / Cell Hypoxia / Glutaredoxins - genetics / Glutaredoxins - metabolism / Glutathione - metabolism / HEK293 Cells / Hindlimb - blood supply / Hindlimb - metabolism / Hindlimb - pathology / Humans / Hypoxia-Inducible Factor 1, alpha Subunit - genetics / Hypoxia-Inducible Factor 1, alpha Subunit - metabolism / Ischemia - genetics / Ischemia - metabolism / Ischemia - pathology / Medical Sciences / Mice / Mice, Knockout / Muscle, Skeletal - blood supply / Muscle, Skeletal - metabolism / Muscle, Skeletal - pathology / Protein Stability / Vascular Endothelial Growth Factor A - biosynthesis / Vascular Endothelial Growth Factor A - genetics