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

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 muscle

Regulation of neovascularization by S-glutathionylation via the Wnt5a/sFlt-1 pathway

S-glutathionylation occurs when reactive oxygen or nitrogen species react with protein-cysteine thiols. Glutaredoxin-1 (Glrx) is a cytosolic enzyme which enzymatically catalyses the reduction in S-glutathionylation, conferring reversible signalling function to proteins with redox-sensitive thiols. Glrx can regulate vascular hypertrophy and inflammation by regulating the activity of nuclear factor κB (NF-κB) and actin polymerization. Vascular endothelial growth factor (VEGF)-induced endothelial cell (EC) migration is inhibited by Glrx overexpression. In mice overexpressing Glrx, blood flow recovery, exercise function and capillary density were significantly attenuated after hindlimb ischaemia (HLI). Wnt5a and soluble Fms-like tyrosine kinase-1 (sFlt-1) were enhanced in the ischaemic-limb muscle and plasma respectively from Glrx transgenic (TG) mice. A Wnt5a/sFlt-1 pathway had been described in myeloid cells controlling retinal blood vessel development. Interestingly, a Wnt5a/sFlt-1 pathway was found also to play a role in EC to inhibit network formation. S-glutathionylation of NF-κB components inhibits its activation. Up-regulated Glrx stimulated the Wnt5a/sFlt-1 pathway through enhancing NF-κB signalling. These studies show a novel role for Glrx in post-ischaemic neovascularization, which could define a potential target for therapy of impaired angiogenesis in pathological conditions including diabetes

Author's personal copy Mitofusins are required for angiogenic function and modulate different signaling pathways in cultured endothelial cells

The mitofusin proteins MFN1 and MFN2 function to maintain mitochondrial networks by binding one another and initiating outer mitochondrial membrane fusion. While it has recently been recognized that vascular endothelial cells rely upon mitochondria as signaling rather than energy-producing moieties, the role of mitochondrial dynamics in endothelial cell function has not been addressed. To begin to understand what role mitochondrial dynamics play in this context, we examined the regulation of MFN1 and MFN2 and the consequences of siRNA-mediated knockdown of these proteins in cultured endothelial cells. Treatment with VEGF-A led to the upregulation of MFN2 and, to a lesser extent, MFN1. Knockdown of either MFN led to disrupted mitochondrial networks and diminished mitochondrial membrane potential. Knockdown of either MFN decreased VEGF-mediated migration and differentiation into network structures. MFN ablation also diminished endothelial cell viability and increased apoptosis under low mitogen conditions. Knockdown of MFN2 uniquely resulted in a decrease in the generation of reactive oxygen species as well as the blunting of the gene expression of components of the respiratory chain and transcription factors associated with oxidative metabolism. In contrast, ablation of MFN1 led to the selective reduction of VEGF-stimulated Akt-eNOS signaling. Taken together, our data indicate that mitochondrial dynamics, particularly those mediated by the mitofusins, play a role in endothelial cell function and viability

Endomembrane HRas controls vascular endothelial growth factor-induced nitric oxide synthase-mediated endothelial cell migration

We demonstrate for the first time that endomembrane-delimited H-Ras mediates VEGF-induced activation of endothelial nitric-oxide synthase (eNOS) and migratory response of human endothelial cells. Using thiol labeling strategies and immunofluorescent cell staining, we found that only 31% of total H-Ras is S-palmitoylated, tethering the small GTPase to the plasma membrane but leaving the function of the large majority of endomembrane-localized H-Ras unexplained. Knockdown of H-Ras blocked VEGF-induced PI3K-dependent Akt (Ser-473) and eNOS (Ser-1177) phosphorylation and nitric oxide-dependent cell migration, demonstrating the essential role of H-Ras. Activation of endogenous H-Ras led to recruitment and phosphorylation of eNOS at endomembranes. The loss of migratory response in cells lacking endogenous H-Ras was fully restored by modest overexpression of an endomembrane-delimited H-Ras palmitoylation mutant. These studies define a newly recognized role for endomembrane-localized H-Ras in mediating nitric oxide-dependent proangiogenic signaling

Surfactant-Induced Artifacts during Proteomic Sample Preparation

Bottom-up proteomics is a powerful tool for characterization of protein post-translational modifications (PTMs), where PTMs are identified at the peptide level by mass spectrometry (MS) following protein digestion. However, enzymatic digestion is associated with additional sample processing steps that may potentially introduce artifactual modifications. Here, during an MS study of the PTMs of the regulator of G-protein signaling 4, we discovered that the use of ProteaseMAX, which is an acid-labile surfactant commonly used to improve protein solubilization and digestion efficiency, can lead to <i>in vitro</i> modifications on cysteine residues. These hydrophobic modifications resemble S-palmitoylation and hydroxyfarnesylation, thus discouraging the use of ProteaseMAX in studies of lipid modifications of proteins. Furthermore, since they target the cysteine thiol group, the presence of these artifacts will inevitably lead to inaccuracies in quantitative analysis of cysteine modifications

Administration of Glutaredoxin-1 Attenuates Liver Fibrosis Caused by Aging and Non-Alcoholic Steatohepatitis

Liver fibrosis is a sign of non-alcoholic fatty liver disease progression towards steatohepatitis (NASH) and cirrhosis and is accelerated by aging. Glutaredoxin-1 (Glrx) controls redox signaling by reversing protein S-glutathionylation, induced by oxidative stress, and its deletion causes fatty liver in mice. Although Glrx regulates various pathways, including metabolism and apoptosis, the impact of Glrx on liver fibrosis has not been studied. Therefore, we evaluated the role of Glrx in liver fibrosis induced by aging or by a high-fat, high-fructose diet. We found that: (1) upregulation of Glrx expression level inhibits age-induced hepatic apoptosis and liver fibrosis. In vitro studies indicate that Glrx regulates Fas-induced apoptosis in hepatocytes; (2) diet-induced NASH leads to reduced expression of Glrx and higher levels of S-glutathionylated proteins in the liver. In the NASH model, hepatocyte-specific adeno-associated virus-mediated Glrx overexpression (AAV-Hep-Glrx) suppresses fibrosis and apoptosis and improves liver function; (3) AAV-Hep-Glrx significantly inhibits transcription of Zbtb16 and negatively regulates immune pathways in the NASH liver. In conclusion, the upregulation of Glrx is a potential therapeutic for the reversal of NASH progression by attenuating inflammatory and fibrotic processes