Summary The methionine sulfoxide reductase system has been implicated in aging and protection against oxidative stress. This conserved system reverses the oxidation of methionine residues within proteins. We analyzed one of the components of this system, the methionine sulfoxide reductase A gene, in Caenorhabditis elegans. We found that the msra‐1 gene is expressed in most tissues, particularly in the intestine and the nervous system. Worms carrying a deletion of the msra‐1 gene are more sensitive to oxidative stress, show chemotaxis and locomotory defects, and a 30% decrease in median survival. We established that msra‐1 expression decreases during aging and is regulated by the DAF‐16/FOXO3a transcription factor. The absence of this enzyme decreases median survival and affects oxidative stress resistance of long lived daf‐2 worms. A similar effect of MSRA‐1 absence in wild‐type and daf‐2 (where most antioxidant enzymes are activated) backgrounds, suggests that the lack of this member of the methionine repair system cannot be compensated by the general antioxidant response. Moreover, FOXO3a directly activates the human MsrA promoter in a cell culture system, implying that this could be a conserved mechanism of MsrA regulation. Our results suggest that repair of oxidative damage in proteins influences the rate at which tissues age. This repair mechanism, rather than the general decreased of radical oxygen species levels, could be one of the main determinants of organisms’ lifespan.

Rationale Endothelial adherens junction proteins constitute an important element in the control of microvascular permeability. Platelet-activating factor (PAF) increases permeability to macromolecules via translocation of eNOS to cytosol and stimulation of eNOS-derived NO signaling cascade. The mechanisms by which NO signaling regulates permeability at adherens junctions are still incompletely understood. Objective We explored the hypothesis that PAF stimulates hyperpermeability via S-nitrosation (SNO) of adherens junction proteins. Methods and Results We measured PAF-stimulated S-nitrosation of β-catenin and p120-catenin (p120) in three cell lines: ECV-eNOSGFP, EAhy926 (derived from human umbilical vein) and CVEC (derived from bovine heart endothelium) and in the mouse cremaster muscle in vivo. SNO correlated with diminished abundance of β-catenin and p120 at the adherens junction and with hyperpermeability. TNF-α increased NO production and caused similar increase in S-nitrosation as PAF. To ascertain the importance of eNOS subcellular location in this process, we used ECV-304 cells transfected with cytosolic eNOS (GFPeNOSG2A) and plasma membrane eNOS (GFPeNOSCAAX). PAF induced S-nitrosation of β-catenin and p120 and significantly diminished association between these proteins in cells with cytosolic eNOS but not in cells wherein eNOS is anchored to the cell membrane. Inhibitors of NO production and of S-nitrosation blocked PAF-induced S-nitrosation and hyperpermeability whereas inhibition of the cGMP pathway had no effect. Mass spectrometry analysis of purified p120 identified cysteine 579 as the main S-nitrosated residue in the region that putatively interacts with VE-cadherin. Conclusions Our results demonstrate that agonist-induced SNO contributes to junctional membrane protein changes that enhance endothelial permeability.

Approaches to reduce excessive edema due to the microvascular hyperpermeability that occurs during ischemia-reperfusion (I/R) are needed to prevent muscle compartment syndrome. We tested the hypothesis that cAMP-activated mechanisms actively restore barrier integrity in postischemic striated muscle. We found, using I/R in intact muscles and hypoxia-reoxygenation (H/R, an I/R mimic) in human microvascular endothelial cells (HMVECs), that hyperpermeability can be deactivated by increasing cAMP levels through application of forskolin. This effect was seen whether or not the hyperpermeability was accompanied by increased mRNA expression of VEGF, which occurred only after 4 h of ischemia. We found that cAMP increases in HMVECs after H/R, suggesting that cAMP-mediated restoration of barrier function is a physiological mechanism. We explored the mechanisms underlying this effect of cAMP. We found that exchange protein activated by cAMP 1 (Epac1), a downstream effector of cAMP that stimulates Rap1 to enhance cell adhesion, was activated only at or after reoxygenation. Thus, when Rap1 was depleted by small interfering RNA, H/R-induced hyperpermeability persisted even when forskolin was applied. We demonstrate that) VEGF mRNA expression is not involved in hyperpermeability after brief ischemia, ) elevation of cAMP concentration at reperfusion deactivates hyperpermeability, and) cAMP activates the Epac1-Rap1 pathway to restore normal microvascular permeability. Our data support the novel concepts that ) different hyperpermeability mechanisms operate after brief and prolonged ischemia and) cAMP concentration elevation during reperfusion contributes to deactivation of I/R-induced hyperpermeability through the Epac-Rap1 pathway. Endothelial cAMP management at reperfusion may be therapeutic in I/R injury. Here, we demonstrate that ) stimulation of cAMP production deactivates ischemia-reperfusion-induced hyperpermeability in muscle and endothelial cells;) VEGF mRNA expression is not enhanced by brief ischemia, suggesting that VEGF mechanisms do not activate immediate postischemic hyperpermeability; and ) deactivation mechanisms operate via cAMP-exchange protein activated by cAMP 1-Rap1 to restore integrity of the endothelial barrier.

Nitric oxide (NO) is a key factor in inflammation produced by endothelial nitric oxide synthase (eNOS) in endothelium, whose activity increases after stimulation with pro-inflammatory cytokines. NO activates the soluble guanylate cyclase-protein kinase G and S-nitrosylation (NO modification of free-thiol cysteines in proteins) pathways. NO is classically described as a negative regulator of leukocyte adhesion to endothelial cells. However, agonists activating NO production induce fast leukocyte adhesion suggesting that NO might positively regulate leukocyte adhesion. We tested the hypothesis that eNOS-induced NO promotes leukocyte adhesion through the S-nitrosylation pathway. We stimulated leukocyte adhesion to endothelium using tumor necrosis factor alpha (TNF-α) as pro-inflammatory agonist. ICAM-1 changes were evaluated by biochemical and imaging techniques. Protein kinase C zeta (PKCζ) activity and S-nitrosylation were evaluated by western-blot and biotin switch methods, respectively. TNF-α, at short times of stimulation, activated the eNOS- S-nitrosylation pathway and caused leukocyte adhesion to endothelial cells in vivo and in vitro. TNF-α induced NO led to changes in ICAM-1 at the cell surface, which are characteristic of clustering. TNF-α induced NO also produced S-nitrosylation and phosphorylation of PKCζ, association of PKCζ with ICAM-1 and ICAM-1 phosphorylation. The inhibition of PKCz blocked leukocyte adhesion induced by TNF-α. Mass spectrometry analysis of PKCζ identified cysteine 503 as the S-nitrosylated residue in the kinase domain of the protein. Our results reveal a new eNOS-S-nitrosylation mechanism that induces leukocyte adhesion suggesting that PKCζ--S-nitrosylation might be an important regulatory step in early leukocyte adhesion.