eNOS S-glutathionylation Research Articles

KLF2 regulates eNOS uncoupling via Nrf2/HO-1 in endothelial cells under hypoxia and reoxygenation

Kruppel-like factor 2 (KLF2) regulates endothelial functions by modulating endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) pathway. Tetrahydrobiopterin (BH4) and S-glutathionylation of eNOS play essential roles in eNOS uncoupling and activation. However, the influence of KLF2 on eNOS uncoupling and the mechanism of eNOS activation still remain unknown. A hypoxia and reoxygenation (H/R) model of human umbilical vein endothelial cells (HUVECs) was utilized in this study. Cell viability and the eNOS uncoupling-related oxidative stress index were measured. The Nrf2 inhibitor ML385 and HO-1 siRNA were used to elucidate the mechanism of activation. The results show that overexpression of KLF2 increased the cell viability, reduced the lactate dehydrogenase leakage rate, downregulated the generation of O2•− and ONOO−, and increased NO levels and eNOS activity. Overexpression of KLF2 also increased the BH4/BH2 ratio and the GSH/GSSG ratio, thus significantly improving eNOS uncoupling in the H/R model. KLF2 has no regulatory effect on the upstream-associated proteins in eNOS activation. However, when combined with the Nrf2 inhibitor or HO-1 siRNA, the regulatory effect of KLF2 on eNOS uncoupling was strongly reduced. These results suggest that KLF2 could improve eNOS uncoupling via Nrf2/HO-1 in H/R-induced endothelial injury.

High glutathionylation of placental endothelial nitric oxide synthase in preeclampsia

Decreased nitric oxide (NO) bioavailability plays a critical role in the pathophysiology of preeclampsia (PE). Recent evidence indicates that S-glutathionylation may occur on the endothelial nitric oxide synthase (eNOS), leading to eNOS uncoupling, characterized by a decreased NO production and an increased generation of superoxide anion (O2•–). We hypothesized that eNOS glutathionylation may occur in PE placentas and participate in eNOS dysfunction.The glutathionylation of eNOS was investigated in thirteen PE-affected patients and in nine normal pregnancies. Immunofluorescence, confocal microscopy and western-blot experiments carried out on eNOS immunoprecipitates, revealed a high level of eNOS glutathionylation in PE placentas, mostly reversed by dithiotreitol (DTT), thus indicative of S-glutathionylation. In order to investigate whether eNOS glutathionylation may alter trophoblast migration, an important event occurring during early placentation, cultured HTR-8/SVneo human trophoblasts (HTR8) were exposed either to low pO2 (O2 1%) or to pO2 changes (O2 1–20%), in order to generate oxidative stress. Trophoblasts exposed to low pO2, did not undergo oxidative stress nor eNOS S-glutathionylation, and were able to generate NO and migrate in a wound closure model. In contrast, trophoblasts submitted to low/high pO2 changes, exhibited oxidative stress and a (DTT reversible) S-glutathionylation of eNOS, associated with reduced NO production and migration. The autonomous production of NO seemed necessary for the migratory potential of HTR8, as suggested by the inhibitory effect of eNOS silencing by small interfering RNAs, and the eNOS inhibitor L-NAME, in low pO2 conditions. Finally, the addition of the NO donor, NOC-18 (5 µM), restored in part the migration of HTR8, thereby emphasizing the role of NO in trophoblast homeostasis.In conclusion, the high level of eNOS S-glutathionylation in PE placentas provides new insights in the mechanism of eNOS dysfunction in this disease.

Exercise Training Has Contrasting Effects in Myocardial Infarction and Pressure Overload Due to Divergent Endothelial Nitric Oxide Synthase Regulation.

The beneficial effects of exercise training (EX) on cardiac pathology are well recognized. Previously, we found that the effects of EX on cardiac dysfunction in mice critically depend on the underlying etiology. EX exerted beneficial effects after myocardial infarction (MI); however, cardiac pathology following pressure overload produced by transverse aortic constriction (TAC) was aggravated by EX. In the presented study, we investigated whether the contrasting effects of EX on cardiac dysfunction can be explained by an etiology-specific response of endothelial nitric oxide (NO) synthase (eNOS) to EX, which divergently affects the balance between nitric oxide and superoxide. For this purpose, mice were exposed to eight weeks of voluntary wheel running or sedentary housing (SED), immediately after sham, MI, or TAC surgery. Left ventricular (LV) function was assessed using echocardiography and hemodynamic measurements. EX ameliorated LV dysfunction and remodeling after MI, but not following TAC, in which EX even aggravated fibrosis. Strikingly, EX attenuated superoxide levels after MI, but exacerbated NOS-dependent superoxide levels following TAC. Similarly, elevated eNOS S-glutathionylation and eNOS monomerization, which were observed in both MI and TAC, were corrected by EX in MI, but aggravated by EX after TAC. Additionally, EX reduced antioxidant activity in TAC, while it was maintained following EX in MI. In conclusion, the present study shows that EX mitigates cardiac dysfunction after MI, likely by attenuating eNOS uncoupling-mediated oxidative stress, whereas EX tends to aggravate cardiac dysfunction following TAC, likely due to exacerbating eNOS-mediated oxidative stress.

Role of glutathione biosynthesis in endothelial dysfunction and fibrosis

Glutathione (GSH) biosynthesis is essential for cellular redox homeostasis and antioxidant defense. The rate-limiting step requires glutamate-cysteine ligase (GCL), which is composed of the catalytic (GCLc) and the modulatory (GCLm) subunits. To evaluate the contribution of GCLc to endothelial function we generated an endothelial-specific Gclc haplo-insufficient mouse model (Gclc e/+ mice). In murine lung endothelial cells (MLEC) derived from these mice we observed a 50% reduction in GCLc levels compared to lung fibroblasts from the same mice. MLEC obtained from haplo-insufficient mice showed significant reduction in GSH levels as well as increased basal and stimulated ROS levels, reduced phosphorylation of eNOS (Ser 1177) and increased eNOS S-glutathionylation, compared to MLEC from wild type (WT) mice. Studies in mesenteric arteries demonstrated impaired endothelium-dependent vasodilation in Gclc(e/+) male mice, which was corrected by pre-incubation with GSH-ethyl-ester and BH4. To study the contribution of endothelial GSH synthesis to renal fibrosis we employed the unilateral ureteral obstruction model in WT and Gclc(e/+) mice. We observed that obstructed kidneys from Gclc(e/+) mice exhibited increased deposition of fibrotic markers and reduced Nrf2 levels. We conclude that the preservation of endothelial GSH biosynthesis is not only critical for endothelial function but also in anti-fibrotic responses.

OP-16 - Role of Glutathione Biosynthesis in Endothelial Dysfunction and Fibrosis

Glutathione (GSH) biosynthesis is essential for cellular redox homeostasis and antioxidant defence. The rate-limiting step requires glutathione cysteine ligase (GCL), which is composed of the catalytic (GCLc) and modulatory (GCLm) subunits. To evaluate the contribution of GCLc to endothelial function we generated endothelial-specific GCLc haploinsufficient (GCLc e/+ mice). In murine lung endothelial cells (MLEC) derived from these mice we corroborated a 50% reduction in GCLc levels compared to fibroblasts from the same mice. MLEC obtained from haploinsufficient mice showed significant reduction in GSH levels as well as increased basal and stimulated ROS levels, reduced phosphorylation of eNOS (Ser 1176) and increased eNOS S-glutathionylation, compared to MLEC from wild type (WT) mice. Studies in mesenteric arteries demonstrated impaired endothelium-dependent vasodilation in GCLc e/+ male mice, which was corrected by pre-incubation with ethyl-ester GSH and BH4. To study the contribution of endothelial GSH synthesis to renal fibrosis we employed the unilateral ureteral obstruction model in WT and GCLc e/+ mice. We observed that obstructed kidneys from GCLc e/+ mice exhibited increased deposition of fibrotic markers and reduced Nrf2 levels. We conclude that the preservation of endothelial GSH biosynthesis is critical for endothelial function and anti-fibrotic response.

Fecal microbiota transplantation (FMT) could reverse the severity of experimental necrotizing enterocolitis (NEC) via oxidative stress modulation

Fecal microbiota transplantation (FMT) has been used successfully to treat a variety of gastroenterological diseases. The alterations of microbiota in mouse models of necrotizing enterocolitis (NEC) as well as in patients suggested the possibility of treating NEC with FMT. Here we show that FMT caused an improvement in the histopathology and symptoms of NEC in WT mice, but not Grx1-/- mice. FMT eliminated O2•– production and promoted NO production in experimental NEC mice though the modulation of S-glutathionylation of eNOS (eNOS-SSG). FMT decreased the extent of TLR4-mediated proinflammatory signaling though TLR9 in the intestinal mucosa tissue. FMT also suppressed intestinal apoptosis and bacterial translocation across the intestinal barrier, which was accompanied by decreased inflammatory cytokine levels, altered bacterial microbiota, and regulated lymphocyte proportions. FMT is effective in a mouse model of NEC through the modulation of oxidative stress and reduced colon inflammation.

Combined l-citrulline and glutathione administration prevents neuronal cell death following transient brain ischemia

We previously reported that oral l-citrulline (l-Cit) administration antagonizes neuronal cell death in hippocampus following transient brain ischemia and that oral glutathione (GSH) administration prevents neuronal death through antioxidant activity. Here, we tested potential synergy of combined l-Cit and GSH administration in protection against neuronal death following cerebral ischemia. One day after a 20-min bilateral common carotid artery occlusion (BCCAO), mice were orally administered l-Cit or GSH alone (at 40 or 100mg/kgp.o.) or both (at 40mg/kgp.o. each) daily for 10days. The combination, but not l-Cit or GSH alone at 40mg/kgp.o., significantly prevented neuronal death in the hippocampal CA1 region in BCCAO mice. Consistently, combined l-Cit and GSH administration improved memory-related behavioral deficits observed in BCCAO mice. Combination treatment also significantly rescued reduced endothelial nitric oxide synthase (eNOS) protein levels and antagonized eNOS S-glutathionylation seen following BCCAO ischemia. Recovery of eNOS activity was confirmed by in vivo NO production in hippocampus of BCCAO mice. Taken together, combined administration of l-Cit with GSH rescues eNOS function, thereby inhibiting delayed neuronal death in hippocampus.

Implication of eNOS Uncoupling in Cardiovascular Disease

Under physiological conditions, nitric oxide (NO) is produced in the vasculature mainly by the endothelial nitric oxide synthase (eNOS). Endothelial NO relaxes blood vessels, inhibits platelet activity, and protects against atherosclerosis. Under pathological conditions such as hypertension, diabetes, and hypercholesterolemia, eNOS may become uncoupled. Uncoupled eNOS generates superoxide at the expense of NO and contributes substantially to oxidative stress and endothelial dysfunction. Major mechanisms of eNOS uncoupling include deficiency of the eNOS cofactor tetrahydrobiopterin, deficiency of the eNOS substrate L-arginine, and eNOS S-glutathionylation. Reversal of eNOS uncoupling may represent a feasible strategy for the prevention and treatment of cardiovascular diseases.

75 - VEGFR2 Signaling Driven by Cellular Redox Status

Angiogenesis (a process of new vessel formation) is triggered by tissue ischemia. Paradoxically, angiogenesis is diminished in cardiovascular diseases (CVDs) due to a decrease in cellular glutathione (GSH) and increase in reactive oxygen species (ROS). Interestingly, our laboratory has demonstrated that a small decrease in cellular GSH promotes vascular endothelial growth factor (VEGF)-driven ischemic angiogenesis while a larger decrease in cellular GSH does not. Thus, we hypothesize VEGF receptor2 (VEGFR2) activation is regulated by intracellular GSH redox state. To specifically study the role of altered GSH redox state on angiogenesis, we used diamide to oxidize GSH to GSSG in human aortic endothelial cells. GSSG can react with protein cysteine thiols to form S-glutathionylated proteins that in turn affect protein function and stability. Our preliminary experiments show that drastic changes in GSH/GSSG activate VEGFR2 independent of VEGF while a moderate change does not activate the receptor. Interestingly, the addition of H2O2 alone did not activate VEGFR2 suggesting a role for GSH/GSSG in regulating VEGFR2. To understand the mechanism of S-glutathionylation in VEGFR2 activation, we used dimedone, a sulfenic acid trap. Pretreatment with dimedone abolished VEGFR2 activation after diamide treatment suggesting that cysteine sulfenic acid formation precedes S-glutathionylation. Local ROS production in the vicinity of proteins can effectively mediate cellular signaling. On that note, O2●– derived from endothelial nitric oxide synthase (eNOS) could activate VEGFR2 in altered GSH/GSSG. S-glutathionylation of eNOS leads to its uncoupling and drives O2●– generation. To test the role of O2●– in VEGFR2 activation in altered GSH redox state, we inhibited eNOS using L-NAME or siRNA and then treated with diamide. VEGFR2 activation decreased after eNOS inhibition suggesting a potential role for eNOS in VEGFR2 activation under altered GSH redox state. We believe a small decrease in GSH/GSSG would result in priming of VEGFR2 signaling pathway while a larger decrease in GSH/GSSG will result in dysfunctional signaling. In conclusion, the GSH/GSSH ratio is important in triggering and promoting VEGFR2 signaling.

Contribution of glutaredoxin-1 to S-glutathionylation of endothelial nitric oxide synthase for mesenteric nitric oxide generation in experimental necrotizing enterocolitis

Endothelial nitric oxide synthase (eNOS) is critical for intestinal microcirculatory perfusion and therefore plays a key role in the development of necrotizing enterocolitis (NEC). eNOS-derived nitric oxide (NO) is inhibited by S-glutathionylation of eNOS (eNOS-SSG), which can be reversed by glutaredoxin-1 (Grx1). Therefore, the objective of this study was to investigate the interplay between Grx1 and eNOS in regulating the following inflammation signal during the development of NEC. Primary mouse intestinal microvascular endothelial cells (MIMECs) and peritoneal macrophages were subjected to lipopolysaccharide treatment, and Grx1-/- mice were subjected to an NEC-inducing regimen of formula feeding in combination with hypoxia and hypothermia. The eNOS-SSG level and its activity were assessed using immunoprecipitated assay and NO production evaluation. NO-mediated Toll-like receptor 4 (TLR4) signaling and inflammation injury were further defined. NEC severity was significantly increased in Grx1-/- mice. Grx1-/- mice with NEC showed significantly decreased NO and increased O2•- production with increases in eNOS-SSG. Furthermore, TLR4 signaling, which is required for the development of NEC, was enhanced in the Grx1-deficient mice. These results suggest that eNOS-SSG within the MIMECs inhibited NO production and enhanced TLR4 activity, which were implicated in the pathogenesis of NEC. Grx1 deficiency increases the severity of NEC in association with eNOS-SSG.

Abstract 14119: Contrasting Effects of Exercise Training After Acute Myocardial Infarction versus Aortic Stenosis Depend Critically on the Regulation of Endothelial Nitric Oxide Synthase

Introduction: The cardiovascular benefits of exercise training (EX) are widely appreciated. Previously we found that the cardiac effects of EX critically depend on the underlying cause of heart disease. Hypothesis: The underlying etiology determines how EX affects the endothelial nitric oxide (NO) synthase (eNOS)-mediated balance between NO and superoxide (O2-). Methods: Mice were subjected to sham surgery, myocardial infarction (MI) or transverse aortic constriction (TAC), and subsequently exposed to 8 weeks of voluntary wheel running or sedentary housing. Left ventricular (LV) function was assessed by echocardiography and hemodynamic measurements; fibrosis by Picro-sirius Red staining; peroxynitrite (ONOO-) and O2- production by luminol- and lucigenin-enhanced chemiluminescence respectively, with or without the NOS inhibitor L-NAME; eNOS uncoupling and eNOS S-glutathionylation by western blot and coimmunoprecipitation, respectively; cardiac NO by the Griess reaction. Results: EX ameliorated LV dysfunction and fibrosis in MI but not TAC (Table 1). Strikingly, O2- generation was blunted by EX in MI, but exacerbated by EX in TAC, which was largely NOS-dependent. Accordingly, eNOS uncoupling and eNOS S-glutathionylation were corrected by EX in MI but aggravated in TAC mice. In parallel, ONOO- levels was attenuated by EX in MI but aggravated by EX in TAC. Cardiac NO levels were reduced in MI and TAC and normalized by EX in MI. Conclusions: The contrasting effects of EX in MI vs TAC can be explained by the highly divergent effects of EX on eNOS regulation, resulting in blunted vs aggravated oxidative stress by EX in MI vs TAC.

Normal and high eNOS levels are detrimental in both mild and severe cardiac pressure-overload

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) exerts beneficial effects in a variety of cardiovascular disease states. Studies on the benefit of eNOS activity in pressure-overload cardiac hypertrophy and dysfunction produced by aortic stenosis are equivocal, which may be due to different expression levels of eNOS or different severities of pressure-overload. Consequently, we investigated the effects of eNOS-expression level on cardiac hypertrophy and dysfunction produced by mild or severe pressure-overload. To unravel the impact of eNOS on pressure-overload cardiac dysfunction we subjected eNOS deficient, wildtype and eNOS overexpressing transgenic (eNOS-Tg) mice to 8weeks of mild or severe transverse aortic constriction (TAC) and studied cardiac geometry and function at the whole organ and tissue level. In both mild and severe TAC, lack of eNOS ameliorated, whereas eNOS overexpression aggravated, TAC-induced cardiac remodeling and dysfunction. Moreover, the detrimental effects of eNOS in severe TAC were associated with aggravation of TAC-induced NOS-dependent oxidative stress and by further elevation of eNOS monomer levels, consistent with enhanced eNOS uncoupling. In the presence of TAC, scavenging of reactive oxygen species with N-acetylcysteine reduced eNOS S-glutathionylation, eNOS monomer and NOS-dependent superoxide levels in eNOS-Tg mice to wildtype levels. Accordingly, N-acetylcysteine improved cardiac function in eNOS-Tg but not in wildtype mice with TAC. In conclusion, independent of the severity of TAC, eNOS aggravates cardiac remodeling and dysfunction, which appears due to TAC-induced eNOS uncoupling and superoxide production.

Abstract 15954: Both Tetrahydrobiopterin Depletion and eNOS S-Glutathionytion Contribute to eNOS Uncoupling in Coronary Disease Patients

Background: Vascular endothelial dysfunction (VED) is associated with increased oxidative stress due to eNOS uncoupling with increased superoxide (•O2-) production and has important prognostic clinical implications for subsequent cardiovascular events. Our recent studies in hypertensive rats revealed a novel redox-regulated pathway through which eNOS is uncoupled due to S-glutathionylation. However, its role in human vascular disease remains unclear. Hence, we studied eNOS S-glutathionylation and uncoupling and in aortic tissue from patients undergoing cardiovascular surgery and heart transplantation (N=25). Methods: Studies were performed using vessels from spontaneously hypertensive rats (SHRs), control WKY rats and aortic tissue from patients undergoing cardiovascular surgery. S-glutathionylation was determined with anti-glutathione and anti-eNOS, and an antibody specific for eNOS S-glutathionylation at C689 (anti-eNOS Cys689-SG). •O2- was measured using fluorescent probe DHE in the absence/presence of the NOS inhibitor, L-NAME, DTT, or the SOD mimetic, MnTBAP. The NOS cofactor, BH4, was measured by HPLC. Results & Conclusion: S-glutathionylation of eNOS (eNOS-SG) was evident by immunohistology in SHR vessels compared to respective WKY control rat vessels. Similarly, immune assays of the aortic samples from vascular disease patients (VD) showed an increase in eNOS-SG protein when compared to no-disease control tissues (ND) and increased •O2- production was detected. Addition of DTT or β-ME decreased the levels of eNOS-SG with decreased •O2- generation. BH4 content was markedly decreased in the vessels of patients with coronary disease. BH4:BH2 ratio was decreased by over 5-fold also contributing to eNOS uncoupling. This provides the first evidence for an association of both eNOS S-glutathionylation and BH4 depletion leading to eNOS uncoupling with loss of endothelial dependent vasodilation in vessels of coronary disease patients.

Abstract 13082: On-Pump Cardiac Surgery in Humans Induces Myocardial Nitric Oxide Synthase Dysfunction via S-Glutathionylation of eNOS

Background and Aims: The mechanism responsible for LV dysfunction after cardiac surgery is only partially understood. In isolated rat hearts subjected to a prolonged ischemia-reperfusion protocol, LV dysfunction was associated with uncoupling of endothelial nitric oxide synthase (eNOS) activity secondary to oxidation of the NOS co-factor, tetrahydrobiopterin (BH4). Here we investigated the effect of cardiopulmonary bypass (CPB) and reperfusion on myocardial NOS activity and ROS production in 116 patients who underwent elective cardiac surgery. Results: Paired samples of the right atrial appendages (RAA) were obtained prior to venous cannulation of the right atrium (PRE) and after myocardial reperfusion (POST). Atrial superoxide (O2-) release increased significantly after reperfusion (in RLU/sec/mg: from 37.83 ± 3.71 to 65.02 ± 6.01 in the POST samples; p < 0.001, n = 46 samples from 23 patients) due to uncoupling of NOS activity (D-NAME - L-NAME = ΔO2- in RLU/sec/mg: from - 31.47 ± 9.15 PRE to 40.75 ± 11.84 POST; p < 0.001 in n = 36) and increased mitochondrial and NOX2 oxidase activity (by 309% and 149%, respectively, P<0.001). POST atrial content of BH4 was reduced by 32% (P=0.001), as was the activity of the rate-limiting enzyme in BH4 synthesis, GTP-cyclohydrolase 1 (GTPCH1, by 50%, P<0.001). NOS activity (HPLC detection of 14C L-Arginine to L-Citrulline conversion) decreased significantly after reperfusion (by 60%; p < 0.001) whereas the protein content of NOS isoforms remained unchanged. Surprisingly, the PRE-POST difference in NOS activity was not affected by BH4 supplementation (10 μM) or NOX2 inhibition with the gp-91-ds tat peptide. Instead, we identified increased eNOS s-glutathionylation as the main mechanism responsible for NOS uncoupling after reperfusion. Reversing NOS s-glutathionylation with dithiothreitol (DTT: 100 μM; n=18) completely restored NOS activity after reperfusion (p =0.34). Conclusions: Our findings indicate that NOS s-glutathionylation, rather than BH4 depletion, accounts for NOS dysfunction in patients after cardiac surgery and CPB. Although myocardial NOX2 activity is increased after reperfusion, NOX2 do not appear to be the source of oxidative stress responsible for NOS dysfunction under these conditions.

Nox2-dependent glutathionylation of endothelial NOS leads to uncoupled superoxide production and endothelial barrier dysfunction in acute lung injury.

Microvascular barrier integrity is dependent on bioavailable nitric oxide (NO) produced locally by endothelial NO synthase (eNOS). Under conditions of limited substrate or cofactor availability or by enzymatic modification, eNOS may become uncoupled, producing superoxide in lieu of NO. This study was designed to investigate how eNOS-dependent superoxide production contributes to endothelial barrier dysfunction in inflammatory lung injury and its regulation. C57BL/6J mice were challenged with intratracheal LPS. Bronchoalveolar lavage fluid was analyzed for protein accumulation, and lung tissue homogenate was assayed for endothelial NOS content and function. Human lung microvascular endothelial cell (HLMVEC) monolayers were exposed to LPS in vitro, and barrier integrity and superoxide production were measured. Biopterin species were quantified, and coimmunoprecipitation (Co-IP) assays were performed to identify protein interactions with eNOS that putatively drive uncoupling. Mice exposed to LPS demonstrated eNOS-dependent increased alveolar permeability without evidence for altered canonical NO signaling. LPS-induced superoxide production and permeability in HLMVEC were inhibited by the NOS inhibitor nitro-l-arginine methyl ester, eNOS-targeted siRNA, the eNOS cofactor tetrahydrobiopterin, and superoxide dismutase. Co-IP indicated that LPS stimulated the association of eNOS with NADPH oxidase 2 (Nox2), which correlated with augmented eNOS S-glutathionylation both in vitro and in vivo. In vitro, Nox2-specific inhibition prevented LPS-induced eNOS modification and increases in both superoxide production and permeability. These data indicate that eNOS uncoupling contributes to superoxide production and barrier dysfunction in the lung microvasculature after exposure to LPS. Furthermore, the results implicate Nox2-mediated eNOS-S-glutathionylation as a mechanism underlying LPS-induced eNOS uncoupling in the lung microvasculature.

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.

Carbamylated low-density lipoprotein induces endothelial dysfunction

Cardiovascular events remain the leading cause of death in Western world. Atherosclerosis is the most common underlying complication driven by low-density lipoproteins (LDL) disturbing vascular integrity. Carbamylation of lysine residues, occurring primarily in the presence of chronic kidney disease (CKD), may affect functional properties of lipoproteins; however, its effect on endothelial function is unknown. Low-density lipoprotein from healthy donors was isolated and carbamylated. Vascular reactivity after treatment with native LDL (nLDL) or carbamylated LDL (cLDL) was examined in organ chambers for isometric tension recording using aortic rings of wild-type or lectin-like-oxidized LDL receptor-1 (LOX-1) transgenic mice. Reactive oxygen species (ROS) and nitric oxide (NO) production were determined using electron spin resonance spectroscopy. The effect of LDL-carbamyl-lysine levels on cardiovascular outcomes was determined in patients with CKD during a median follow-up of 4.7 years. Carbamylated LDL impaired endothelium-dependent relaxation to acetylcholine or calcium-ionophore A23187, but not endothelium-independent relaxation to sodium nitroprusside. In contrast, nLDL had no effect. Carbamylated LDL enhanced aortic ROS production by activating NADPH-oxidase. Carbamylated LDL stimulated endothelial NO synthase (eNOS) uncoupling at least partially by promoting S-glutathionylation of eNOS. Carbamylated LDL-induced endothelial dysfunction was enhanced in LOX-1 transgenic mice. In patients with CKD, LDL-carbamyl-lysine levels were significant predictors for cardiovascular events and all-cause mortality. Carbamylation of LDL induces endothelial dysfunction via LOX-1 activation and increased ROS production leading to eNOS uncoupling. This indicates a novel mechanism in the pathogenesis of atherosclerotic disease which may be pathogenic and prognostic in patients with CKD and high plasma levels of cLDL.

PSS235 - eNOS S-Glutathionylation Mediated by Peroxynitrite: Mechanism of Endothelial Disfunction

PSS235 - eNOS S-Glutathionylation Mediated by Peroxynitrite: Mechanism of Endothelial Disfunction

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.

Molecular Mechanisms of the Crosstalk Between Mitochondria and NADPH Oxidase Through Reactive Oxygen Species—Studies in White Blood Cells and in Animal Models

Oxidative stress is involved in the development of cardiovascular disease. There is a growing body of evidence for a crosstalk between different enzymatic sources of oxidative stress. With the present study, we sought to determine the underlying crosstalk mechanisms, the role of the mitochondrial permeability transition pore (mPTP), and its link to endothelial dysfunction. NADPH oxidase (Nox) activation (oxidative burst and translocation of cytosolic Nox subunits) was observed in response to mitochondrial reactive oxygen species (mtROS) formation in human leukocytes. In vitro, mtROS-induced Nox activation was prevented by inhibitors of the mPTP, protein kinase C, tyrosine kinase cSrc, Nox itself, or an intracellular calcium chelator and was absent in leukocytes with p47phox deficiency (regulates Nox2) or with cyclophilin D deficiency (regulates mPTP). In contrast, the crosstalk in leukocytes was amplified by mitochondrial superoxide dismutase (type 2) (MnSOD(+/-)) deficiency. In vivo, increases in blood pressure, degree of endothelial dysfunction, endothelial nitric oxide synthase (eNOS) dysregulation/uncoupling (e.g., eNOS S-glutathionylation) or Nox activity, p47phox phosphorylation in response to angiotensin-II (AT-II) in vivo treatment, or the aging process were more pronounced in MnSOD(+/-) mice as compared with untreated controls and improved by mPTP inhibition by cyclophilin D deficiency or sanglifehrin A therapy. These results provide new mechanistic insights into what extent mtROS trigger Nox activation in phagocytes and cardiovascular tissue, leading to endothelial dysfunction. Our data show that mtROS trigger the activation of phagocytic and cardiovascular NADPH oxidases, which may have fundamental implications for immune cell activation and development of AT-II-induced hypertension.