Bacterial Pyocyanin Inducible Keratin 6A Accelerates Closure of Epithelial Defect under Conditions of Mitochondrial Dysfunction.

Repair of epithelial defect is complicated by infection and related metabolites. Pyocyanin (PYO) is one such metabolite that is secreted during Pseudomonas aeruginosa infection. Keratinocyte (KC) migration is required for the closure of skin epithelial defects. This work sought to understand PYO-KC interaction and its significance in tissue repair. Stable Isotope Labeling by Amino acids in Cell culture proteomics identified mitochondrial dysfunction as the top pathway responsive to PYO exposure in human KCs. Consistently, functional studies showed mitochondrial stress, depletion of reducing equivalents, and adenosine triphosphate. Strikingly, despite all stated earlier, PYO markedly accelerated KC migration. Investigation of underlying mechanisms revealed, to our knowledge, a previously unreported function of keratin 6A in KCs. Keratin 6A was PYO inducible and accelerated closure of epithelial defect. Acceleration of closure was associated with poor quality healing, including compromised expression of apical junction proteins. This work recognizes keratin 6A for its role in enhancing KC migration under conditions of threat posed by PYO. Qualitatively deficient junctional proteins under conditions of defensive acceleration of KC migration explain why an infected wound close with deficient skin barrier function as previously reported.

Cytoglobin has potent superoxide dismutase function.

Cytoglobin (Cygb) was discovered as a novel type of globin that is expressed in mammals; however, its functions remain uncertain. While Cygb protects against oxidant stress, the basis for this is unclear, and the effect of Cygb on superoxide metabolism is unknown. From dose-dependent studies of the effect of Cygb on superoxide catabolism, we identify that Cygb has potent superoxide dismutase (SOD) function. Initial assays using cytochrome c showed that Cygb exhibits a high rate of superoxide dismutation on the order of 108 M-1 ⋅ s-1 Spin-trapping studies also demonstrated that the rate of Cygb-mediated superoxide dismutation (1.6 × 108 M-1 ⋅ s-1) was only ∼10-fold less than Cu,Zn-SOD. Stopped-flow experiments confirmed that Cygb rapidly dismutates superoxide with rates within an order of magnitude of Cu,Zn-SOD or Mn-SOD. The SOD function of Cygb was inhibited by cyanide and CO that coordinate to Fe3+-Cygb and Fe2+-Cygb, respectively, suggesting that dismutation involves iron redox cycling, and this was confirmed by spectrophotometric titrations. In control smooth-muscle cells and cells with siRNA-mediated Cygb knockdown subjected to extracellular superoxide stress from xanthine/xanthine oxidase or intracellular superoxide stress triggered by the uncoupler, menadione, Cygb had a prominent role in superoxide metabolism and protected against superoxide-mediated death. Similar experiments in vessels showed higher levels of superoxide in Cygb-/- mice than wild type. Thus, Cygb has potent SOD function and can rapidly dismutate superoxide in cells, conferring protection against oxidant injury. In view of its ubiquitous cellular expression at micromolar concentrations in smooth-muscle and other cells, Cygb can play an important role in cellular superoxide metabolism.

Defining the reducing system of the NO dioxygenase cytoglobin in vascular smooth muscle cells and its critical role in regulating cellular NO decay.

In smooth muscle, cytoglobin (Cygb) functions as a potent nitric oxide (NO) dioxygenase and regulates NO metabolism and vascular tone. Major questions remain regarding which cellular reducing systems regulate Cygb-mediated NO metabolism. To better define the Cygb-mediated NO dioxygenation process in vascular smooth muscle cells (SMCs), and the requisite reducing systems that regulate cellular NO decay, we assessed the intracellular concentrations of Cygb and its putative reducing systems and examined their roles in the process of NO decay. Cygb and the reducing systems, cytochrome b5 (B5)/cytochrome b5 reductase (B5R) and cytochrome P450 reductase (CPR) were measured in aortic SMCs. Intracellular Cygb concentration was estimated as 3.5 µM, while B5R, B5, and CPR were 0.88, 0.38, and 0.15 µM, respectively. NO decay in SMCs was measured following bolus addition of NO to air-equilibrated cells. siRNA-mediated knockdown experiments indicated that âˆ¼78% of NO metabolism in SMCs is Cygb-dependent. Of this, ∼87% was B5R- and B5-dependent. CPR knockdown resulted in a small decrease in the NO dioxygenation rate (VNO), while depletion of ascorbate had no effect. Kinetic analysis of VNO for the B5/B5R/Cygb system with variation of B5 or B5R concentrations from their SMC levels showed that VNO exhibits apparent Michaelis-Menten behavior for B5 and B5R. In contrast, linear variation was seen with change in Cygb concentration. Overall, B5/B5R was demonstrated to be the major reducing system supporting Cygb-mediated NO metabolism in SMCs with changes in cellular B5/B5R levels modulating the process of NO decay.

The novel SOD mimetic GC4419 increases cancer cell killing with sensitization to ionizing radiation while protecting normal cells.

While radiotherapy is a widely used treatment for many types of human cancer, problems of radio-resistance and side effects remain. Side effects induced by ionizing radiation (IR) arise primarily from its propensity to trigger inflammation and oxidative stress with damage of normal cells and tissues near the treatment area. The highly potent superoxide dismutase mimetic, GC4419 (Galera Therapeutics), rapidly enters cells and is highly effective in dismutating superoxide (O2•-). We performed studies to assess the potency of GC4419 in cancer killing and radio-sensitization in human lung cancer cells and normal immortalized lung cells. Treatment with GC4419 did not alter the radical generation during IR, primarily hydroxyl radical (.OH); however, it quenched the increased levels of O2•- detected in the cancer cells before and following IR. GC4419 triggered cancer cell death and inhibited cancer cell proliferation with no adverse effect on normal cells. Combination of GC4419 with IR augmented the cytotoxic effects of IR on cancer cells compared to monotherapy, while protecting normal cells from IR-induced cell death. DNA fragmentation and caspase-3 activity assays showed that combination of GC4419 with IR enhances cancer cell apoptosis. Moreover, GC4419 increased IR-induced Bax levels with decreased Bcl-2 and elevated Bax/Bcl-2 ratio following treatment. GC4419 increased TrxR activity in the normal cells but decreased activity in cancer cells, conferring increased cancer cell sensitivity to oxidative stress. In conclusion, GC4419 increases the cytotoxic and pro-apoptotic activity of IR in lung cancer cells while decreasing injury in normal cells.

Chronic cigarette smoke exposure triggers a vicious cycle of leukocyte and endothelial-mediated oxidant stress that results in vascular dysfunction.

Although there is a strong association between cigarette smoking exposure (CSE) and vascular endothelial dysfunction (VED), the underlying mechanisms by which CSE triggers VED remain unclear. Therefore, studies were performed to define these mechanisms using a chronic mouse model of cigarette smoking (CS)-induced cardiovascular disease mirroring that in humans. C57BL/6 male mice were subjected to CSE for up to 48 wk. CSE impaired acetylcholine (ACh)-induced relaxation of aortic and mesenteric segments and triggered hypertension, with mean arterial blood pressure at 32 and 48 wk of exposure of 122 ± 6 and 135 ± 5 mmHg compared with 99 ± 4 and 102 ± 6 mmHg, respectively, in air-exposed mice. CSE led to monocyte activation with superoxide generation in blood exiting the pulmonary circulation. Macrophage infiltration with concomitant increase in NADPH oxidase subunits p22phox and gp91phox was seen in aortas of CS-exposed mice at 16 wk, with further increase out to 48 wk. Associated with this, increased superoxide production was detected that decreased with Nox inhibition. Tetrahydrobiopterin was progressively depleted in CS-exposed mice but not in air-exposed controls, resulting in endothelial nitric oxide synthase (eNOS) uncoupling and secondary superoxide generation. CSE led to a time-dependent decrease in eNOS and Akt expression and phosphorylation. Overall, CSE induces vascular monocyte infiltration with increased NADPH oxidase-mediated reactive oxygen species generation and depletes the eNOS cofactor tetrahydrobiopterin, uncoupling eNOS and triggering a vicious cycle of oxidative stress with VED and hypertension. Our study provides important insights toward understanding the process by which smoking contributes to the genesis of cardiovascular disease and identifies biomarkers predictive of disease.NEW & NOTEWORTHY In a chronic model of smoking-induced cardiovascular disease, we define underlying mechanisms of smoking-induced vascular endothelial dysfunction (VED). Smoking exposure triggered VED and hypertension and led to vascular macrophage infiltration with concomitant increase in superoxide and NADPH oxidase levels as early as 16 wk of exposure. This oxidative stress was accompanied by tetrahydrobiopterin depletion, resulting in endothelial nitric oxide synthase uncoupling with further superoxide generation triggering a vicious cycle of oxidative stress and VED.

Characterization of CD38 in the major cell types of the heart: endothelial cells highly express CD38 with activation by hypoxia-reoxygenation triggering NAD(P)H depletion.

The NAD(P)+-hydrolyzing enzyme CD38 is activated in the heart during the process of ischemia and reperfusion, triggering NAD(P)(H) depletion. However, the presence and role of CD38 in the major cell types of the heart are unknown. Therefore, we characterize the presence and function of CD38 in cardiac myocytes, endothelial cells, and fibroblasts. To comprehensively evaluate CD38 in these cells, we measured gene transcription via mRNA, as well as protein expression and enzymatic activity. Endothelial cells strongly expressed CD38, while only low expression was present in cardiac myocytes with intermediate levels in fibroblasts. In view of this high level expression in endothelial cells and the proposed role of CD38 in the pathogenesis of endothelial dysfunction, endothelial cells were subjected to hypoxia-reoxygenation to characterize the effect of this stress on CD38 expression and activity. An activity-based CD38 imaging method and CD38 activity assays were used to characterize CD38 activity in normoxic and hypoxic-reoxygenated endothelial cells, with marked CD38 activation seen following hypoxia-reoxygenation. To test the impact of hypoxia-reoxygenation-induced CD38 activation on endothelial cells, NAD(P)(H) levels and endothelial nitric oxide synthase (eNOS)-derived NO production were measured. Marked NADP(H) depletion with loss of NO and increase in superoxide production occurred following hypoxia-reoxygenation that was prevented by CD38 inhibition or knockdown. Thus, endothelial cells have high expression of CD38 which is activated by hypoxia-reoxygenation triggering CD38-mediated NADP(H) depletion with loss of eNOS-mediated NO generation and increased eNOS uncoupling. This demonstrates the importance of CD38 in the endothelium and explains the basis by which CD38 triggers post-ischemic endothelial dysfunction.

Oxygen binding and nitric oxide dioxygenase activity of cytoglobin are altered to different extents by cysteine modification.

Cytoglobin (Cygb), like other members of the globin family, is a nitric oxide (NO) dioxygenase, metabolizing NO in an oxygen (O2)-dependent manner. We examined the effect of modification of cysteine sulfhydryl groups of Cygb on its O2 binding and NO dioxygenase activity. The two cysteine sulfhydryls of Cygb were modified to form either an intramolecular disulfide bond (Cygb_SS), thioether bonds to N-ethylmaleimide (NEM; Cygb_SC), or were maintained as free SH groups (Cygb_SH). It was observed that the NO dioxygenase activity of Cygb only slightly changed (~ 25%) while the P50 of O2 binding to Cygb changed over four-fold with these modifications. Our results suggest that it is possible to separately regulate one Cygb function (such as O2 binding) without largely affecting the other Cygb functions (such as its NO dioxygenase activity).

Cytoglobin regulates blood pressure and vascular tone through nitric oxide metabolism in the vascular wall.

The identity of the specific nitric oxide dioxygenase (NOD) that serves as the main in vivo regulator of O2-dependent NO degradation in smooth muscle remains elusive. Cytoglobin (Cygb) is a recently discovered globin expressed in fibroblasts and smooth muscle cells with unknown function. Cygb, coupled with a cellular reducing system, efficiently regulates the rate of NO consumption by metabolizing NO in an O2-dependent manner with decreased NO consumption in physiological hypoxia. Here we show that Cygb is a major regulator of NO degradation and cardiovascular tone. Knockout of Cygb greatly prolongs NO decay, increases vascular relaxation, and lowers blood pressure and systemic vascular resistance. We further demonstrate that downregulation of Cygb prevents angiotensin-mediated hypertension. Thus, Cygb has a critical role in the regulation of vascular tone and disease. We suggest that modulation of the expression and NOD activity of Cygb represents a strategy for the treatment of cardiovascular disease.

Luteolinidin Protects the Postischemic Heart through CD38 Inhibition with Preservation of NAD(P)(H).

We recently showed that ischemia/reperfusion (I/R) of the heart causes CD38 activation with resultant depletion of the cardiac NADP(H) pool, which is most marked in the endothelium. This NADP(H) depletion was shown to limit the production of nitric oxide by endothelial nitric oxide synthase (eNOS), which requires NADPH for nitric oxide production, resulting in greatly altered endothelial function. Therefore, intervention with CD38 inhibitors could reverse postischemic eNOS-mediated endothelial dysfunction. Here, we evaluated the potency of the CD38 inhibitor luteolinidin, an anthocyanidin, at blocking CD38 activity and preserving endothelial and myocardial function in the postischemic heart. Initially, we characterized luteolinidin as a CD38 inhibitor in vitro to determine its potency and mechanism of inhibition. We then tested luteolinidin in the ex vivo isolated heart model, where we determined luteolinidin uptake with aqueous and liposomal delivery methods. Optimal delivery methods were then further tested to determine the effect of luteolinidin on postischemic NAD(P)(H) and tetrahydrobiopterin levels. Finally, through nitric oxide synthase-dependent coronary flow and left ventricular functional measurements, we evaluated the efficacy of luteolinidin to protect vascular and contractile function, respectively, after I/R. With enhanced postischemic preservation of NADPH and tetrahydrobiopterin, there was a dose-dependent effect of luteolinidin on increasing recovery of endothelium-dependent vasodilatory function, as well as enhancing the recovery of left ventricular contractile function with increased myocardial salvage. Thus, luteolinidin is a potent CD38 inhibitor that protects the heart against I/R injury with preservation of eNOS function and prevention of endothelial dysfunction.

Trityl radicals in perfluorocarbon emulsions as stable, sensitive, and biocompatible oximetry probes.

EPR oximetry with the use of trityl radicals can enable sensitive O2 measurement in biological cells and tissues. However, in vitro cellular and in vivo biological applications are limited by rapid trityl probe degradation or biological clearance and the need to enhance probe O2 sensitivity. We synthesized novel perfluorocarbon (PFC) emulsions, ∼200nm droplet size, containing esterified perchlorinated triphenyl methyl (PTM) radicals dispersed in physiological aqueous buffers. These formulations exhibit excellent EPR signal stability, over 20-fold greater than free PTM probes, with high oxygen sensitivity ∼17mG/mmHg enabling pO2 measurement in aqueous solutions or cell suspensions with sensitivity >0.5mmHg. Thus, PFC-PTM probes hold great promise to enable combined O2 delivery and sensing as needed to restore or enhance tissue oxygenation in disease.

Nitrones reverse hyperglycemia-induced endothelial dysfunction in bovine aortic endothelial cells.

Hyperglycemia has been implicated in the development of endothelial dysfunction through heightened ROS production. Since nitrones reverse endothelial nitric oxide synthase (eNOS) dysfunction, increase antioxidant enzyme activity, and suppress pro-apoptotic signaling pathway and mitochondrial dysfunction from ROS-induced toxicity, the objective of this study was to determine whether nitrone spin traps DMPO, PBN and PBN-LA were effective at duplicating these effects and improving glucose uptake in an in vitro model of hyperglycemia-induced dysfunction using bovine aortic endothelial cells (BAEC). BAEC were cultured in DMEM medium with low (5.5mM glucose, LG) or high glucose (50mM, HG) for 14 days to model in vivo hyperglycemia as experienced in humans with metabolic disease. Improvements in cell viability, intracellular oxidative stress, NO and tetrahydrobiopterin (BH4)​ levels, mitochondrial membrane potential, glucose transport, and activity of antioxidant enzymes were measured from single treatment of BAEC with nitrones for 24h after hyperglycemia. Chronic hyperglycemia significantly increased intracellular ROS by 50%, decreased cell viability by 25%, reduced NO bioavailability by 50%, and decreased (BH4) levels by 15% thereby decreasing NO production. Intracellular glucose transport and superoxide dismutase (SOD) activity were also decreased by 50% and 25% respectively. Nitrone (PBN and DMPO, 50 µM) treatment of BAEC grown in hyperglycemic conditions resulted in the normalization of outcome measures except for SOD and catalase activities. Our findings demonstrate that the nitrones reverse the deleterious effects of hyperglycemia in BAEC. We believe that in vivo testing of these nitrone compounds in models of cardiometabolic disease is warranted.

Sulfite Oxidase Activity of Cytochrome c: Role of Hydrogen Peroxide.

In humans, sulfite is generated endogenously by the metabolism of sulfur containing amino acids such as methionine and cysteine. Sulfite is also formed from exposure to sulfur dioxide, one of the major environmental pollutants. Sulfite is used as an antioxidant and preservative in dried fruits, vegetables, and beverages such as wine. Sulfite is also used as a stabilizer in many drugs. Sulfite toxicity has been associated with allergic reactions characterized by sulfite sensitivity, asthma, and anaphylactic shock. Sulfite is also toxic to neurons and cardiovascular cells. Recent studies suggest that the cytotoxicity of sulfite is mediated by free radicals; however, molecular mechanisms involved in sulfite toxicity are not fully understood. Cytochrome c (cyt c) is known to participate in mitochondrial respiration and has antioxidant and peroxidase activities. Studies were performed to understand the related mechanism of oxidation of sulfite and radical generation by ferric cytochrome c (Fe3+ cyt c) in the absence and presence of H2O2. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with sulfite, Fe3+ cyt c, and H2O2. An EPR spectrum corresponding to the sulfite radical adducts of DMPO (DMPO-SO3-) was obtained. The amount of DMPO-SO3- formed from the oxidation of sulfite by the Fe3+ cyt c increased with sulfite concentration. In addition, the amount of DMPO-SO3- formed by the peroxidase activity of Fe3+ cyt c also increased with sulfite and H2O2 concentration. From these results, we propose a mechanism in which the Fe3+ cyt c and its peroxidase activity oxidizes sulfite to sulfite radical. Our results suggest that Fe3+ cyt c could have a novel role in the deleterious effects of sulfite in biological systems due to increased production of sulfite radical. It also shows that the increased production of sulfite radical may be responsible for neurotoxicity and some of the injuries which occur to humans born with molybdenum cofactor and sulfite oxidase deficiencies.

Depletion of NADP(H) due to CD38 activation triggers endothelial dysfunction in the postischemic heart.

In the postischemic heart, coronary vasodilation is impaired due to loss of endothelial nitric oxide synthase (eNOS) function. Although the eNOS cofactor tetrahydrobiopterin (BH4) is depleted, its repletion only partially restores eNOS-mediated coronary vasodilation, indicating that other critical factors trigger endothelial dysfunction. Therefore, studies were performed to characterize the unidentified factor(s) that trigger endothelial dysfunction in the postischemic heart. We observed that depletion of the eNOS substrate NADPH occurs in the postischemic heart with near total depletion from the endothelium, triggering impaired eNOS function and limiting BH4 rescue through NADPH-dependent salvage pathways. In isolated rat hearts subjected to 30 min of ischemia and reperfusion (I/R), depletion of the NADP(H) pool occurred and was most marked in the endothelium, with >85% depletion. Repletion of NADPH after I/R increased NOS-dependent coronary flow well above that with BH4 alone. With combined NADPH and BH4 repletion, full restoration of NOS-dependent coronary flow occurred. Profound endothelial NADPH depletion was identified to be due to marked activation of the NAD(P)ase-activity of CD38 and could be prevented by inhibition or specific knockdown of this protein. Depletion of the NADPH precursor, NADP(+), coincided with formation of 2'-phospho-ADP ribose, a CD38-derived signaling molecule. Inhibition of CD38 prevented NADP(H) depletion and preserved endothelium-dependent relaxation and NO generation with increased recovery of contractile function and decreased infarction in the postischemic heart. Thus, CD38 activation is an important cause of postischemic endothelial dysfunction and presents a novel therapeutic target for prevention of this dysfunction in unstable coronary syndromes.

Genetic and hypoxic alterations of the microRNA-210-ISCU1/2 axis promote iron-sulfur deficiency and pulmonary hypertension.

Iron-sulfur (Fe-S) clusters are essential for mitochondrial metabolism, but their regulation in pulmonary hypertension (PH) remains enigmatic. We demonstrate that alterations of the miR-210-ISCU1/2 axis cause Fe-S deficiencies in vivo and promote PH. In pulmonary vascular cells and particularly endothelium, hypoxic induction of miR-210 and repression of the miR-210 targets ISCU1/2 down-regulated Fe-S levels. In mouse and human vascular and endothelial tissue affected by PH, miR-210 was elevated accompanied by decreased ISCU1/2 and Fe-S integrity. In mice, miR-210 repressed ISCU1/2 and promoted PH. Mice deficient in miR-210, via genetic/pharmacologic means or via an endothelial-specific manner, displayed increased ISCU1/2 and were resistant to Fe-S-dependent pathophenotypes and PH. Similar to hypoxia or miR-210 overexpression, ISCU1/2 knockdown also promoted PH. Finally, cardiopulmonary exercise testing of a woman with homozygous ISCU mutations revealed exercise-induced pulmonary vascular dysfunction. Thus, driven by acquired (hypoxia) or genetic causes, the miR-210-ISCU1/2 regulatory axis is a pathogenic lynchpin causing Fe-S deficiency and PH. These findings carry broad translational implications for defining the metabolic origins of PH and potentially other metabolic diseases sharing similar underpinnings.

Silver-zinc redox-coupled electroceutical wound dressing disrupts bacterial biofilm.

Pseudomonas aeruginosa biofilm is commonly associated with chronic wound infection. A FDA approved wireless electroceutical dressing (WED), which in the presence of conductive wound exudate gets activated to generate electric field (0.3-0.9V), was investigated for its anti-biofilm properties. Growth of pathogenic P. aeruginosa strain PAO1 in LB media was markedly arrested in the presence of the WED. Scanning electron microscopy demonstrated that WED markedly disrupted biofilm integrity in a setting where silver dressing was ineffective. Biofilm thickness and number of live bacterial cells were decreased in the presence of WED. Quorum sensing genes lasR and rhlR and activity of electric field sensitive enzyme, glycerol-3-phosphate dehydrogenase was also repressed by WED. This work provides first electron paramagnetic resonance spectroscopy evidence demonstrating that WED serves as a spontaneous source of reactive oxygen species. Redox-sensitive multidrug efflux systems mexAB and mexEF were repressed by WED. Taken together, these observations provide first evidence supporting the anti-biofilm properties of WED.

Effect of temperature, pH and heme ligands on the reduction of Cygb(Fe(3+)) by ascorbate.

Cytoglobin (Cygb) plays a role in regulating vasodilation in response to changes in local oxygen concentration by altering the rate of nitric oxide (NO) metabolism. Because the reduction of Cygb(Fe(3+)) by a reductant is the control step for Cygb-mediated NO metabolism, we examined the effects of temperature, pH, and heme ligands on the Cygb(Fe(3+)) reduction by ascorbate (Asc) under anaerobic conditions. The standard enthalpy of Cygb(Fe(3+)) reduction by Asc was determined to be 42.4 ± 3.1 kJ/mol. The rate of Cygb(Fe(3+)) reduction increased ~6% per °C when temperature varied from 35°C to 40°C. The yield and the rate of Cygb(Fe(3+)) reduction significantly increases with pH (2-3 times per pH unit), paralleling the formation of the Asc ion (A(2-)) and the increased stability of reduced state of heme iron at high pH values. Heme ligand cyanide (CN(-)) decreased the yield and the rate of Cygb(Fe(3+)) reduction, but ligands CO and NO allowed the process of Cygb(Fe(3+)) reduction to continue to completion. Critical information is provided for modeling and prediction of the process of Cygb-mediated NO metabolism in vessels in a range of temperature and pH values.

Hypoxia and reoxygenation induce endothelial nitric oxide synthase uncoupling in endothelial cells through tetrahydrobiopterin depletion and S-glutathionylation.

Ischemia-reperfusion injury is accompanied by endothelial hypoxia and reoxygenation that trigger oxidative stress with enhanced superoxide generation and diminished nitric oxide (NO) production leading to endothelial dysfunction. Oxidative depletion of the endothelial NO synthase (eNOS) cofactor tetrahydrobiopterin can trigger eNOS uncoupling, in which the enzyme generates superoxide rather than NO. Recently, it has also been shown that oxidative stress can induce eNOS S-glutathionylation at critical cysteine residues of the reductase site that serves as a redox switch to control eNOS coupling. While superoxide can deplete tetrahydrobiopterin and induce eNOS S-glutathionylation, the extent of and interaction between these processes in the pathogenesis of eNOS dysfunction in endothelial cells following hypoxia and reoxygenation remain unknown. Therefore, studies were performed on endothelial cells subjected to hypoxia and reoxygenation to determine the severity of eNOS uncoupling and the role of cofactor depletion and S-glutathionylation in this process. Hypoxia and reoxygenation of aortic endothelial cells triggered xanthine oxidase-mediated superoxide generation, causing both tetrahydrobiopterin depletion and S-glutathionylation with resultant eNOS uncoupling. Replenishing cells with tetrahydrobiopterin along with increasing intracellular levels of glutathione greatly preserved eNOS activity after hypoxia and reoxygenation, while targeting either mechanism alone only partially ameliorated the decrease in NO. Endothelial oxidative stress, secondary to hypoxia and reoxygenation, uncoupled eNOS with an altered ratio of oxidized to reduced glutathione inducing eNOS S-glutathionylation. These mechanisms triggered by oxidative stress combine to cause eNOS dysfunction with shift of the enzyme from NO to superoxide production. Thus, in endothelial reoxygenation injury, normalization of both tetrahydrobiopterin levels and the glutathione pool are needed for maximal restoration of eNOS function and NO generation.

Redox modulation of endothelial nitric oxide synthase by glutaredoxin-1 through reversible oxidative post-translational modification.

S-Glutathionylation is a redox-regulated modification that uncouples endothelial nitric oxide synthase (eNOS), switching its function from nitric oxide (NO) synthesis to (•)O2(-) generation, and serves to regulate vascular function. While in vitro or in vivo eNOS S-glutathionylation with modification of Cys689 and Cys908 of its reductase domain is triggered by high levels of glutathione disulfide (GSSG) or oxidative thiyl radical formation, it remains unclear how this process may be reversed. Glutaredoxin-1 (Grx1), a cytosolic and glutathione-dependent enzyme, can reverse protein S-glutathionylation; however, its role in regulating eNOS S-glutathionylation remains unknown. We demonstrate that Grx1 in the presence of glutathione (GSH) (1 mM) reverses GSSG-mediated eNOS S-glutathionylation with restoration of NO synthase activity. Because Grx1 also catalyzes protein S-glutathionylation with an increased [GSSG]/[GSH] ratio, we measured its effect on eNOS S-glutathionylation when the [GSSG]/[GSH] ratio was >0.2, which can occur in cells and tissues under oxidative stress, and observed an increased level of eNOS S-glutathionylation with a marked decrease in eNOS activity without uncoupling. This eNOS S-glutathionylation was reversed with a decrease in the [GSSG]/[GSH] ratio to <0.1. Liquid chromatography and tandem mass spectrometry identified a new site of eNOS S-glutathionylation by Grx1 at Cys382, on the surface of the oxygenase domain, without modification of Cys689 or Cys908, each of which is buried within the reductase. Furthermore, Grx1 was demonstrated to be a protein partner of eNOS in vitro and in normal endothelial cells, supporting its role in eNOS redox regulation. In endothelial cells, Grx1 inhibition or gene silencing increased the level of eNOS S-glutathionylation and decreased the level of cellular NO generation. Thus, Grx1 can exert an important role in the redox regulation of eNOS in cells.

Differences in oxygen-dependent nitric oxide metabolism by cytoglobin and myoglobin account for their differing functional roles.

The endogenous vasodilator nitric oxide (NO) is metabolized in tissues in an oxygen-dependent manner. In skeletal and cardiac muscle, high concentrations of myoglobin (Mb) function as a potent NO scavenger. However, the Mb concentration is very low in vascular smooth muscle, where low concentrations of cytoglobin (Cygb) may play a major role in metabolizing NO. Questions remain regarding how low concentrations of Cygb and Mb differ in terms of NO metabolism, and the basis for their different cellular roles and functions. In this study, electrode techniques were used to perform comparative measurements of the kinetics of NO consumption by Mb and Cygb. UV/Vis spectroscopic methods and computer simulations were performed to study the reaction of Mb and Cygb with ascorbate (Asc) and the underlying mechanism. It was observed that the initial rate of Cygb(3+) reduction by Asc was 415-fold greater than that of Mb(3+). In the low [O2] range (0-50 µM), the Cygb-mediated NO consumption rate is ~ 500 times more sensitive to changes in O2 concentration than that of Mb. The reduction of Cygb(3+) by Asc follows a reversible kinetic model, but that of Mb(3+) is irreversible. A reaction mechanism for Cygb(3+) reduction by Asc is proposed, and the reaction equilibrium constants are determined. Our results suggest that the rapid reduction of Cygb by cellular reductants enables Cygb to efficiently regulate NO metabolism in the vascular wall in an oxygen-dependent manner, but the slow rate of Mb reduction does not show this oxygen dependence.

Esterified dendritic TAM radicals with very high stability and enhanced oxygen sensitivity.

In this work, we have developed a new class of dendritic TAM radicals (TG, TdG, and dTdG) through a convergent method based on the TAM core CT-03 or its deuterated analogue dCT-03 and trifurcated Newkome-type monomer. Among these radicals, dTdG exhibits the best EPR properties with sharpest EPR singlet and highest O(2) sensitivity due to deuteration of both the ester linker groups and the TAM core CT-03. Like the previous dendritic TAM radicals, these new compounds also show extremely high stability toward various reactive species owing to the dendritic encapsulation. The highly charged nature of these molecules resulting from nine carboxylate groups prevents concentration-dependent EPR line broadening at physiological pH. Furthermore, we demonstrate that these TAM radicals can be easily derivatized (e.g., PEGylation) at the nine carboxylate groups and the resulting PEGylated analogue dTdG-PEG completely inhibits the albumin binding, thereby enhancing suitability for in vivo applications. These new dendritic TAM radicals show great potential for in vivo EPR oximetric applications and provide insights on approaches to develop improved and targeted EPR oximetric probes for biomedical applications.