Uncoupling of Endothelial Nitric Oxidase Synthase by Hypochlorous Acid
Role of NAD(P)H Oxidase–Derived Superoxide and Peroxynitrite
Objective— The aim of the present study is to determine whether hypochlorous acid (HOCl), the major oxidant of leukocyte-derived myeloperoxidase (MPO), oxidizes the zinc-thiolate center of endothelial nitric oxide synthase (eNOS) and uncouples the enzyme.
Methods and Results— Exposure of purified recombinant eNOS to HOCl (≥100 μmol/L) released zinc and disrupted the enzyme-active eNOS dimers. In parallel with increased detections of both O2·− and ONOO−, clinically relevant concentrations of HOCl disrupted eNOS dimers in cultured human umbilical vein endothelial cells (HUVEC) at concentration 10- to 100-fold lower than those required for recombinant eNOS. In HUVEC, HOCl increased the translocation of both p67phox and p47phox of NAD(P)H oxidase and the phosphorylation of atypical protein kinase C-ζ. Further, genetic or pharmacological inhibition of either NAD(P)H oxidase–derived O2·− or PKC-ζ or NOS abolished the effects of HOCl on eNOS dimers. Consistently, HOCl increased both O2·− and ONOO− and eNOS dimer oxidation in isolated mouse aortas from C57BL/6 but less in those of gp91phox knock-out mice. Finally, in human carotid atherosclerotic arteries, eNOS predominantly existed as monomers in parallel with increased staining of both MPO and 3-nitrotyrosine.
Conclusions— We conclude that HOCl uncouples eNOS by ONOO− generated from PKC-ζ–dependent NAD(P)H oxidase.
Cardiovascular diseases are frequently associated with vascular inflammation even in the early stage, characterized by leukocyte and platelet adherence to the vascular wall.1,2 There is evidence that leukocyte-derived myeloperoxidase (MPO) is a potent inducer for vascular injury. MPO from adhered leukocytes is bound to vascular endothelium3 and is transcytosed to its luminal side4 where it can catalytically consume nitric oxide (NO) to reduce NO bioactivity.5,6 In cardiovascular diseases, MPO is found to be both present and active in human atherosclerotic lesions.7 Under physiological conditions, HOCl is the major product of leukocyte protein MPO.8 HOCl is able to cause protein oxidation. For example, immunostainings for HOCl-modified proteins are shown to be significantly elevated within and outside of endothelial cells of human atherosclerotic lesions,9 suggesting that HOCl-mediated protein oxidation occurs in atherosclerosis. In addition, clinically relevant concentrations of HOCl are shown to induce endothelial cell apoptosis,10 oxidize LDL,11 and impair NO bioavailability.12 However, the targets and mechanism(s) by which HOCl contributes to vascular injury remained elusive.
See page 2585
A key determinant of endothelial biology is the cell redox state, and a key molecule that mediates endothelial function is NO.13,14 NO is a free radical gaseous molecule and is synthesized by the action of the enzymes nitric oxide synthases (NOS). The catalytic mechanisms of NOS involve flavin-mediated electron transport from the C-terminal–bound NADPH to the N-terminal heme center where oxygen is reduced and incorporated into the guanidine group of L-arginine giving rise to NO and L-citrulline. All three NOS require an active dimer to release NO, and regulation of the dimeric state of the enzyme complex has been shown to be an important regulatory step in the function of all NOS isoforms. L-arginine and tetrahydrobiopterin (BH4) are critical factors in maintaining the dimeric state of the enzyme, which is crucial in regulating how electrons are transferred within the enzyme complex. In the setting of limited L-arginine or BH4 levels, the enzyme functions in an “uncoupled” state in which NADPH-derived electrons are added to molecular O2 rather than L-arginine, with O2·− as products. This phenomenon was first recognized in purified nNOS but also extended to eNOS. O2·− generated by eNOS has been implicated in a variety of experimental and clinical vascular disease states including diabetes, cigarette smoking, hypertension, chronic nitrate tolerance, and overt atherosclerosis.15 For example, chronic overexpression of eNOS does not inhibit but accelerates atherosclerosis under hypercholesterolemia, suggesting that eNOS might not release vasoprotective NO but O2·− in vivo.15 How eNOS becomes uncoupled remained poorly understood. The present study was aimed to study how HOCl, the principle oxidant from leukocyte-derived MPO, triggers eNOS oxidation and uncouples the enzyme.
Methods and Materials
Human umbilical vein endothelial cells (HUVECs) and cell culture media were obtained from Cascade Biologics. Polyclonal or monoclonal antibodies against eNOS (Transduction Laboratories), p47phox, and p67phox were obtained from Santa Cruz Biotechnology. The antibody against 3-NT was from the Upstate Biotechnology, Inc. Dihydroethidium (DHE) was purchased from Molecular Probes. Calcium ionophore A23187 was obtained from Sigma Chemical Co. Bovine recombinant eNOS obtained from Cayman Chemical, was further purified using a 2′,5′-ADP-Sepharose 4B column and calmodulin-Sepharose affinity column as described previously.16,17
Human Carotid Artery Atherosclerotic Plaques
Human carotid artery atherosclerotic plaques were obtained from sixteen consecutive, not previously examined, surgical inpatients enlisted to undergo carotid endarterectomy for extracranial high-grade internal carotid artery stenosis (>70% luminal narrowing). The degree of luminal narrowing was determined by repeated Doppler echography and intraarterial cerebral angiography using the criteria of the North American Symptomatic Carotid Endarterectomy Trial (NASCET). Carotid artery endarterectomy was conducted in standard fashion. Once removed, the plaque sample obtained from each patient was divided into two parts, ie, “low stenosis” and “high” stenosis areas, according to the degree of carotid artery stenosis. The samples were immediately either frozen in liquid nitrogen (LN2) or formalin fixed for further analysis. The study was reviewed and approved by the institutional review board of the University of Tennessee Medical Center at Knoxville.
Mice and Aortas Preparation
Female gene knock-out mice (gp91phox−/−) and the genetic controls C57BL/6J mice, 10 weeks of age, were obtained from the Jackson Laboratory (Bar Harbor, Me). Mice were housed in temperature-controlled cages with a 12-hour light-dark cycle and given free access to water and normal chows. Mouse aortas were removed immediately after being euthanized with inhaled isoflurane and cervical dislocation. The animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee.
The isolated aortas were treated with HOCl as reported.12 Briefly, isolated mice aortas were removed, cleared of connective tissue, and immersed in warm PSS (37°C) for equilibration for 90 minutes. Subsequently, vessel segments were incubated for 30 minutes in control PSS or in PSS containing HOCl. After extensive washings with PSS, DHE (10 μmol/L) was then added to the PSS for 30 minutes followed by an additional 30 minutes in control PSS. The segments were subjected to cryotome tissue dissection for immunochemistry (for 3-NT staining) or fluorescent microscopy (for DHE staining) as previously described.18,19 The staining signal was quantified using AlphaEaseFC software (Alpha Innotech) on the image taken under the same amplification and exposure time.
Confluent HUVECs were exposed to HOCl for 15 minutes at concentrations indicated. The residual HOCl was removed by extensive wash with PBS buffer. HOCl-treated HUVEC were further kept in culture media at 37°C for 30 minutes to 2 hours. The concentrations of HOCl were determined spectrophotometrically in 0.1 mol/L NaOH (E292 nm=350 mol/L cm−1).
Assay of eNOS Dimer/Monomer
The total eNOS expression was detected by using Western blots. eNOS dimers and monomers were assayed by using low temperature SDS-PAGE, as described previously.16
Detection of Zinc Release
Zinc release was measured by using PAR assay as described previously.16
Detection of Reactive Oxygen Species
Intracellular O2·− was assessed by using DHE fluorescence. Cells were incubated with DHE (1 μmol/L) for 30 minutes, followed by fluorescent measurement under Em/Ex=480/580 nm using Synergy HT Multi-Detection Microplate Reader (BioTek Instrument, Inc). O2·− generated by recombinant eNOS was assayed by using the DHE fluorescence/high-performance liquid chromatography (HPLC) assay19 but with modification. Briefly, recombinant eNOS (10 μg) was incubated with DHE (0.5 μmol/L) in assay buffer (Tris-HCI buffer, pH 7.4; calmodulin: 1 μg/mL; L-arginine: 0.5 mmol/L) on ice for 30 minutes, followed by methanol extraction. HPLC was applied to separate and quantify oxyethidium (product of DHE with O2·−) and ethidium (product of DHE auto-oxidation) by column C-18 (mobile phase: gradient acetonitrile and 0.1% trifluoroacetic acid).19 O2·− production was determined by the conversion of DHE into oxyethidine.
Separation of Cytosolic and Membrane Fractions
Cytosolic and membrane fractions of cells were prepared as described previously.20
Confluent HUVECs were infected with either adenovirus encoding Cu/Zn SOD (SOD-1), p67phox dominant negative (p67phox-DN), or PKC-ζ dominant negative (PKC-ζ-DN), as described previously.21 Adenovirus encoding green fluorescence protein (GFP) was used as a control.
Assay of Nitric Oxide Synthase Activity
The NO synthesizing activity of eNOS was determined by quantifying the rate of the conversion of [3H] L-arginine to [3H] L-citrulline in the presence or absence of calcium as previously described.22
Assay of Nitric Oxide Bioactivity
NO bioactivity was determined using cyclic GMP (cGMP) ELISA kit from R&D System, Inc.
Results were analyzed with a 2-way ANOVA. Values are expressed as mean±SEM for n assays. A probability value of <0.05 is considered statistically significant.
HOCl Disrupts eNOS Dimers, Releases Zinc, Inhibits the NO-Synthetic Activity, and Induces O2·− Production in Purified Recombinant eNOS
We had demonstrated that ONOO−, formed by O2·− and NO at a diffusion-controlled rate, oxidizes the zinc-thiolate cluster of eNOS and uncouples the enzyme.16 The high reactivity of the zinc-thiolate center with ONOO− is likely attributable to the highest charge-to-atomic radius ratio of zinc, which maintains partial cationic character even in a tetra-coordinate complex like zinc-thiolate clusters of eNOS.16 Thus, anionic oxidants, such as ONOO−, can oxidize the coordinated thiols in the process of releasing zinc and forming disulfide bonds. Under reducing conditions (β-mercaptoethanol), the disulfide bonds between eNOS monomers are broken, which result in dissociation of eNOS dimers into monomer. Thus, the dimer/monomer ratios can be used as a marker for the redox status of eNOS.16
Like ONOO−, hypochlorite (OCl−) is an anionic oxidant. Thus, it was interesting to investigate the effects of HOCl on eNOS dimer stability. To this end, purified human or bovine eNOS were exposed to different concentrations of HOCl (final concentration from 1 μmol/L to 1 mmol/L) for 30 minutes. As shown in Figure 1A (stripped and gray bars), HOCl (100 μmol/L) significantly increased zinc release from both human and bovine eNOS. In parallel, HOCl (≥100 μmol/L) dissociated the eNOS dimers into monomers (Figure 1B) and suppressed the conversion of 3H-arginine into 3H-citrulline (Figure 1A, solid bar). However, low concentrations of HOCl (≤10 μmol/L) had no effect on eNOS (Figure 1A, solid bar). Taken together, these results suggest that HOCl at concentrations higher than 100 μmol/L, is able to disrupt eNOS dimers, release zinc, and uncouple the enzyme.
HOCl Inhibits eNOS Activity and NO Bioavailability in Intact HUVECs
We next determined whether HOCl inhibited A23187-induced NO release. Confluent HUVECs were exposed to HOCl for 15 minutes at concentrations indicated. After that, HUVECs were washed with media and further incubated for 2 hours. At the end of the 2 hour incubation, eNOS activity was assayed by measuring the conversion of 3H-arginine into 3H-citrulline. As shown in Figure 1A (white bars), HOCl dose-dependently inhibited eNOS activity.
We next determined whether HOCl impaired the NO bioavailability, as measured by the cGMP contents. HOCl at 10 μmol/L, which did not alter the basal levels of cGMP, significantly suppressed the A23187-upregulated cGMP production (17.5±1.2 versus 6.0±0.7 pmol/mg protein, vehicle versus HC-treated, n=3).
HOCl Disrupts eNOS Dimers and Enhances O2·− Production in Intact HUVECs
We next determined whether HOCl oxidized eNOS dimers in intact cells. As shown, HOCl (1 to 100 μmol/L) concentrations-dependent dissociated eNOS dimers into monomers under reducing condition (Figure 2A). Interestingly, low concentrations of HOCl (1 to 10 μmol/L), which had no effect on eNOS dimers in purified recombinant eNOS (Figure 1B), significantly reduced the levels of eNOS dimers, as seen by the decreased ratios of dimer/monomer in HUVECs (Figure 2A). In addition, HOCl significantly increased the detection of O2·− (Figure 2B). However, HOCl-enhanced O2·− was abolished by L-NAME (1 mmol/L; Figure 2B), suggesting that O2·− by HOCl was eNOS-dependent. Notably these concentrations of HOCl were at least 10-fold lower than those required in purified enzyme. These results suggest that the effects of low concentrations of HOCl observed in HUVECs were unlikely attributable to its direct effects but might be mediated by (a) factor(s) generated after being exposed to HOCl.
We next established the time-dependence of HOCl on eNOS dimers in HUVECs. Unlike in purified eNOS, the maximal effects of HOCl on eNOS dimers in HUVECs were seen at 2 hour incubation (data not shown), which was in parallel with an inhibition on eNOS activity (Figure 1A, open bar). These results further support that HOCl uncoupled eNOS by releasing (an) endogenous factor(s) in HUVECs.
Dissociation of eNOS Dimers in Cells Exposed to HOCl: Evidence of ONOO−-Induced eNOS Dimer Oxidation in HUVECs
Our previous studies16 had demonstrated that ONOO− generated by high glucose exposure oxidizes eNOS dimers into monomers under reducing condition. We next investigated whether ONOO− was involved in HOCl-enhanced eNOS dimer oxidation. Because ONOO− is formed by simultaneous generation of O2·− and NO,23 inhibition of either O2·− or NO production attenuates the formation of ONOO−. To this end, HUVECs were exposed to HOCl (1 to 10 μmol/L) either in the presence of a NOS inhibitor, L-NAME (1 mmol/L) or in cells overexpressing Cu/ZnSOD (to prevent O2·−). Indeed, either inhibition of NOS with L-NAME or SOD overexpression attenuated HOCl-enhanced eNOS dimer oxidation (Figure 2C). Because neither O2·− nor NO at low concentrations alone cause eNOS dimer oxidation,24,25 these results suggest that ONOO− might be involved in HOCl-induced eNOS oxidation.
Exogenous NO has been reported to induce eNOS dimer dissociation through S-nitrosylation of the zinc-binding cysteine.25 We next determined whether HOCl increased S-nitrosylation of eNOS. Using the same biotin-switch method,26 we found that HOCl (1 μmol/L to 1 mmol/L) did not alter the levels of S-nitrosylated eNOS (data not shown).
HOCl Enhances the Formation of ONOO− in HUVECs
We next determined whether HOCl increased the production of ONOO−. ONOO− has a half-life less than 1 second at pH 7.4 and is unstable.23 Instead, ONOO− is an important oxidant for protein tyrosine nitration, and 3-NT is considered as a “footprint” for ONOO− production.24 Thus, we determined whether HOCl increased the formation of 3-NT in HUVECs. Exposure of HUVECs to HOCl significantly increased the detection of 3-NT-postive proteins (Figure 2D, shown only the major band). In addition, either overexpression of Cu/Zn-SOD or L-NAME significantly reduced the levels of 3-NT in HUVECs, implying that HOCl released ONOO− (Figure 2D).
HOCl Induces NAD(P)H Oxidase-Derived O2·− Anions in HUVECs
Several enzymes including NAD(P)H oxidases, xanthine oxidase, mitochondria, or cyclooxygenases could be the potential sources of O2·− leading to eNOS uncoupling.27 We next determined the sources of O2·− in HOCl-treated HUVECs. As shown in Figure 3A, HOCl significantly enhanced O2·− in HUVEC. Inhibition of mitochondrial respiration with either rotenone (5 μmol/L) or antimycin A (1 mmol/L) did not alter HOCl-enhanced O2·− release (data not shown), excluding mitochondria as the sources of O2·−. In contrast, apocynin (1 μmol/L), a selective inhibitor which blocks NAD(P)H oxidase assembly and then its activity, suppressed HOCl-enhanced O2·− (Figure 3A), suggesting NAD(P)H oxidase might be involved.
Further evidence for NAD(P)H oxidase as the source of O2·− came from genetic inhibition of NAD(P)H oxidase. Adenoviral overexpression of the dominant negative mutant of p67phox (p67phox-DN) abolished HOCl-enhanced O2·− release (Figure 3A) and eNOS dimer dissociation (Figure 3B, upper panel). Taken together, these results suggest that NAD(P)H oxidase was the initial source of O2·−, which resulted in the formation of ONOO− and consequent eNOS uncoupling.
HOCl Enhances Translocation of Cytosolic p47phox and p67phox Into Membrane
NAD(P)H oxidase activation requires the translocation of p67phox and p47phox subunits from cytosolic fractions into membranes.28,29 We next investigated whether HOCl activated NAD(P)H oxidase in HUVECs, by measuring the translocation of its subunits, p47phox and p67phox, from cytosolic into membrane fractions. As shown in Figure 3C (panel left), HOCl significantly increased the amount of both p47phox and p67phox in membrane fractions (Figure 3C), suggesting that HOCl might increase the activation of NAD(P)H oxidase in HUVECs.
HOCl-Activated NADP(H) Oxidase Is PKC-ζ–Dependent
We next determined how HOCl activated NAD(P)H oxidase in HUVEC. PKC-ζ was reported to activate NADPH oxidase.30 We next determined whether inhibition of PKC-ζ attenuated HOCl-induced NADPH oxidase activation. As shown in Figure 3C (panel right), inhibition of PKC-ζ with its pseudosubstrate (PKC-ζ-PS, 10 μmol/L) abolished HOCl-induced translocation of both p47phox and p67phox into membranes, suggesting that HOCl might activate PKC-ζ resulting in the translocation of p47phox and p67phox and activation of NAD(P)H oxidase. Further, HOCl dose-dependently increased the phosphorylation of PKC- ζ (Figure 3D). Moreover, overexpression of PKC-ζ-DN mutant attenuated HOCl-induced eNOS dimer dissociation (Figure 3B, bottom panel). Taken together, these results indicate that HOCl activated the NAD(P)H oxidases by activating PKC-ζ.
Increased Myeloperoxidase Stainings Are Associated With Increased eNOS Oxidation in Human Atherosclerotic Plaques
We further determined whether or not HOCl contributed to the status of eNOS dimers/monomers and oxidant stress in human atherosclerosis. To determine the contributions of HOCl in the development of atherosclerosis, we measured the levels of MPO (the major enzyme releasing HOCl), 3-NT, vascular cell adhesion molecule (VCAM)-1, as well as eNOS dimer/monomers in human carotid atherosclerotic plagues with either low or high stenosis. As shown in Figure 4A, the antibody stainings against human MPO, 3-NT, and VCAM-1 were significantly elevated in the arterial tissues with high stenotic areas, compared with the arterial tissues from low stenosis. In parallel, the amounts of eNOS dimers were significantly reduced, and eNOS predominantly existed as eNOS monomers in the arterial tissues with high stenosis. Instead, eNOS remained as dimers in the arterial tissues with low stenosis (Figure 4B). Theses results suggest that HOCl from MPO might increase oxidant stress and eNOS oxidation in human atherosclerotic tissues.
HOCl Increases O2·− and 3-NT in Isolated Thoracic Aorta From C57BL/6J Control But Not in Those of gp91phox Knock-Out Mice
We next determined whether HOCl oxidized eNOS dimers by activating NAD(P)H oxidases in isolated aortas. gp91phox (NOX-2) is one of the catalytical subunits of NADPH oxidase in vascular tissues, and several analogues of gp91phox such as NOX-1 and NOX-4 have been identified in vascular tissues.27 We first determined whether NAD(P)H oxidase is required for HOCl increased O2·− and ONOO− in isolated mouse aortas ex vivo. To this end, mouse aortas isolated from both wild-type C57BL/6J mice and gp91phox knock-out mice were exposed to either vehicle (PBS buffer) or HOCl at concentrations indicated. As shown in Figure 5A, HOCl significantly increased both O2·− and 3-NT in the wild-type mice but less in those of the gp91phox knock-out mice, suggesting that vascular NAD(P)H oxidase was required for HOCl-enhanced O2·− and ONOO−.
We further assayed whether HOCl dissociated eNOS dimers, and if so, the contribution of endogenous ONOO−. As shown in Figure 5B, HOCl significantly decreased the ratios of eNOS dimers to eNOS monomers, indicating the disruption of the zinc-thiolate center of eNOS. Further, uric acid (50 μmol/L), which is a potent scavenger for ONOO− but not for HOCl, significantly attenuated HOCl-enhanced dissociation of eNOS dimers. Becuase uric acid did not alter the eNOS dimer/monomer ratios in normal tissues (Figure 5B), these results strongly suggest that HOCl oxidizes eNOS by HOCl-enhanced ONOO−. Taken together, these data suggest that HOCl at 1 to 10 μmol/L activated vascular NADPH oxidase to produce O2·− and ONOO− on reaction with NO resulting in eNOS oxidation. Thus, HOCl might serve as a “kindling” oxidant to turn eNOS into an O2·− producing enzyme.
In the present study, we have for the first time presented evidence demonstrating that HOCl, the major oxidant from leukocyte-derived MPO, is a potent inducer for eNOS uncoupling and 3-NT in the development of cardiovascular diseases. We found that high concentrations of HOCl directly oxidize the zinc-thiolate center of eNOS. Most importantly, we found that clinically relevant concentrations of HOCl uncouple eNOS and increases 3-NT via endogenous O2·− and ONOO−, likely via NAD(P)H oxidase stimulation. The key evidence can be summarized as follows: First, the concentrations required for eNOS oxidation in intact HUVEC were 10 times less than those used in recombinant eNOS in vitro; Second, the effect of HOCl required at least 30 minutes preincubation, suggesting an endogenous process was required for HOCl; Third, low concentrations of HOCl increased O2·− release and overexpression of SOD but not catalase (not shown) attenuated HOCl-induced eNOS oxidation in intact cells. These data suggest that O2·− generated by HOCl was required for HOCl-induced eNOS oxidation; Fourth, inhibition of flavin-protein or L-NAME, a nonselective NOS inhibitor, abolished the effects of HOCl, suggesting that NO was required for HOCl-induced eNOS oxidation as well; Fifth, HOCl increased the detection of 3-NT, a hallmark of ONOO−, suggesting that HOCl might oxidize eNOS by releasing endogenous ONOO−; Sixth, HOCl increased both O2·− and 3-NT in isolated aortas of wild-type mice but increased much less in NAD(P)H oxidase gp91phox KO mice, indicating that HOCl activated NADPH oxidase ex vivo. Moreover, HOCl oxidized eNOS dimer in isolated aortas from wild types but significantly less in those of gp91phox KO mice, implying that NAD(P)H oxidase-derived O2·− and ONOO− were the initial sources for eNOS dimer oxidation. Finally, uric acid, a potent scavenger for ONOO−, but not for HOCl, significantly prevented HOCl-induced eNOS oxidation, confirming that HOCl via ONOO− oxidizes eNOS in isolated aortas ex vivo. Therefore, our results suggest that HOCl might disrupt the zinc-thiolate center of eNOS via endogenous reactive nitrogen species, likely ONOO−.
We have further demonstrated that HOCl can upregulate O2·− release by activating NAD(P)H oxidase. This is a novel finding, and the key evidence can be summarized as follows: First, HOCl significantly increased the detection of O2·− in intact HUVECs, and inhibition of NAD(P)H oxidase with apocynin abolished the effects of HOCl on eNOS; Second, overexpression of the NAD(P)H oxidase subunit p67phox-DN suppressed HOCl-induced O2·− and eNOS oxidation; Third, low concentrations of HOCl significantly increased the translocation of the NAD(P)H oxidase p67phox and p47phox; Fourth, HOCl increased O2·− and ONOO− in the aortas obtained from the wild-type animals but less in the gp91phox KO mice, suggesting that NAD(P)H oxidase was involved in HOCl-increased O2·− release.
Increased formation of 3-NT has been found in over hundreds of diseases and is considered the footprint of ONOO− in vivo.31 Recently, this conception has been challenged because HOCl is found to generate 3-NT in the presence of nitrite.32 In addition, MPO, which can catalyze 3-NT in the presence of nitrite and hydrogen peroxide (H2O2), has been considered as a major source of 3-NT in vivo because the levels of 3-NT are significantly reduced in MPO knockout mice.33 In the present study, we have provided compelling evidence that MPO-mediated 3-NT might be ONOO−-dependent. In HUVECs, we found that HOCl significantly increased both O2·− and 3-NT in HUVECs, which were inhibited by either overexpression of SOD or inhibition of NOS with L-NAME. These data strongly suggest that HOCl increased 3-NT via ONOO−. Although MPO is able to generate 3-NT using hydrogen peroxide and nitrite as substrates,32 it is very unlikely that subcultured HUVECs contain MPO. In addition, overexpression of SOD, which increases H2O2 to promote MPO-dependent 3-NT,32 actually decreased instead of increased the HOCl-enhanced 3-NT in HUVECs, suggesting the 3-NT is not likely to be generated by MPO. Other possibilities of 3-NT include a chemical reaction of HOCl with nitrite.32 This reaction requires millimolar concentrations of both HOCl and nitrite, which is unlikely in vivo. Enhanced formation of ONOO− was shown to disrupt eNOS dimers and reduce NO production. Cardiovascular risk factors such as oxidized lipids and hyperglycemia increase O2·− generation in endothelium by several different pathways, and O2·− is known to inactivate NO and produce ONOO−. ONOO− can disrupt eNOS protein dimers through oxidation and displacement of the zinc metal ion. Overall, our data strongly suggest that HOCl might increase 3-NT via ONOO−, both of which could be from the activated NAD(P)H oxidase and the uncoupled eNOS. Thus, decrease in 3-NT in MPO KO mice33 might be attributable to a decrease of HOCl-triggered ONOO− in the MPO-KO mice. Our data strongly suggest that ONOO− is a principle source of 3-NT in vivo.
Taken together, our data strongly suggest that HOCl is an important inducer for vascular endothelial dysfunction, and clinically relevant concentrations of HOCl increase O2·− and ONOO− via vascular NAD(P)H oxidase, which generates a “kindling” oxidant, ONOO−, resulting in eNOS uncoupling and endothelial dysfunction both in vitro and in vivo. Because vascular inflammation as well leukocyte adherence occurs even in the early stage of numerous cardiovascular diseases, our results suggest that HOCl-induced eNOS uncoupling might represent a common pathological pathway in the development of vascular diseases including hypertension, diabetes, ischemic injury, and atherosclerosis.
Sources of Funding
This work was supported by the NIH grants (HL079584, HL080499, and HL074399), a Research Award from the American Diabetes Association, a Research Award from the Juveniles Diabetes Research Foundation, a grant from Oklahoma Center for Science and Technology (OCAST), and the funds from the Paul H. Doris Eaton Travis Chair Funds in Endocrinology of the University of Oklahoma Health Sciences Center.
Original received June 23, 2006; final version accepted September 15, 2006.
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