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【摘要】
Objective— 4-Hydroxy-2-nonenal (4-HNE) is an abundant electrophilic lipid that mediates oxidative stress in endothelium by mechanisms that remain controversial. This study examines the effects of 4-HNE on nitric oxide (NO) and superoxide levels in bovine aorta endothelial cells (BAECs).
Methods and Results— Exposure of BAECs to 4-HNE caused a dose-dependent inhibition of NO that correlated with losses of hsp90 and phosphorylated eNOS-serine1179 but not eNOS protein levels. 4-HNE failed to inhibit NO production in sepiapterin and ascorbate supplemented cells suggesting that tetrahydrobiopterin (BH 4 ) is a limiting factor in non supplemented cells. This was verified by quantification of BH 4 by high-performance liquid chromatography analysis with electrochemical detection and by examining GTP cyclohydrolase I (GTPCH) protein levels and activity all of which were diminished by 4-HNE treatment. Analysis of 2-hydroxyethidium indicated that 4-HNE increased superoxide release in BAECs. The effects of 4-HNE on GTPCH and hsp90 were efficiently counteracted by proteasomal inhibition, indicating that depletion of BH 4 by 4-HNE is attributable to specific mechanisms involving protein degradation.
Conclusions— 4-HNE by altering BH 4 homeostasis mediates eNOS-uncoupling and superoxide generation in BAECs. By also decreasing phosphorylation of eNOS-serine 1179 4-HNE may specifically regulate NO/reactive oxygen species fluxes in the endothelium with important consequences to redox signaling.
4-Hydroxy-2-nonenal (4-HNE) mediates oxidative stress in the endothelium by controversial mechanisms. This study shows that 4-HNE uncouples eNOS by promoting GTPCH degradation by the proteasome. Sepiapterin and to a lesser extent ascorbate counteracted loss of NO and superoxide increase. Preventing eNOS uncoupling may be important in decreasing 4-HNE cytotoxicity.
【关键词】 tetrahydrobiopterin eNOS phosphorylation hydroxyethidium glutathione ascorbate
Introduction
Nitric oxide (NO) plays an essential role in preserving vascular function and health. Animal models of genetic eNOS deficiency have shown that interrupted NO supply affects a variety of functions including blood vessel remodeling, 1,2 permeability, 3 blood flow, 4 endothelial adhesiveness, and blood pressure. 4 Alteration of one or more of these parameters is considered a powerful predictor for the development of cardiovascular disease.
Production of NO from eNOS is regulated by a complex process dependent on optimal L-arginine and tetrahydrobiopterin (BH 4 ) supply. 5 At the cellular level, eNOS activity is regulated by interaction with different proteins such as hsp90, caveolin-1 (scaffolding peptide), and G 12. 6,7 Additionally, posttranslational eNOS modifications by covalent attachment of lipids (myristoylation, palmitoylation) or phosphate groups (serine 1177, serine 635, threonine 495) 8,9 have been shown to effectively modulate NO production in endothelial cells without changes in eNOS protein levels. However, under certain conditions NO production coincides with upregulation of eNOS expression. 10,11 There has been an increasing interest in defining the relative role of each of these modifications in the mechanisms altering NO production in hypercholesterolemic patients. Several risk factors for atherothrombosis including hypercholesterolemia, hypertension, and diabetes also promote vascular oxidant stress. Thus, it has been suggested that reactive oxygen species are important in the pathophysiogical process leading to decreased NO and acute plaque activation in the atherothrombotic mechanism.
Supplementation with BH 4 improves vascular relaxation in hypercholesterolemic animal models and humans. 12,13 Also, BH 4 has been shown to delay plaque formation in the ApoE –/– mouse 14 that presents low BH 4 levels. The detrimental effects of high cholesterol on aortic BH 4 levels were shown in hypercholesterolemic rabbits. 15 In this model, however, supplementation with sepiapterin, a BH 4 precursor, did not ameliorate vasoconstriction suggesting that either the relatively high levels of cholesterol or long period of exposure alters BH 4 metabolic pathways in a fashion beyond improvement through acute supplementation therapy. 16 Until now the exact mechanism disturbing BH 4 metabolism in the hypercholesterolemic vascular wall, however, remains unclear. It has been thought that oxidative stress plays a role, although responses are disparate depending on the oxidative challenge. Increased BH 4 levels have been shown in endothelial cells treated with bolus addition of hydrogen peroxide (H 2 O 2 ), 16 whereas cells treated with a continuous flow of H 2 O 2 (glucose/glucose oxidase) or peroxynitrite and angiotensin II were shown to have low BH 4 levels. 17,18 These responses have been linked to direct oxidation of the cofactor and also to changes in the activity of the enzymes involved in BH 4 synthesis or recycling.
The lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) is increased in hypercholesterolemia and atherosclerotic lesions causing accumulation of 4-HNE-protein adducts. 19 Also, 4-HNE promotes endothelial oxidative stress, 20 endothelial barrier dysfunction, 21,22 and apoptosis. 23,24 The relationship between 4-HNE, endothelial alteration, and variations in NO and eNOS pathway, however, remains unclear. Considering that both lipid peroxidation and loss of NO are critical to atherosclerosis, this work examines 4-HNE actions on eNOS regulatory mechanisms. Here we show that 4-HNE inhibits eNOS activity by modifying endothelial GTPCH and hsp90 resulting in BH 4 depletion and inhibition of phosphorylated eNOS-serine 1179. Also, 4-HNE increased superoxide anion radical (O 2 – ) release from BAECs. Therefore, 4-HNE likely increases ROS and oxidative stress in endothelial cells by disturbing eNOS regulatory mechanisms. This mechanism is likely relevant to alterations in endothelial functions in atherosclerosis.
Methods
Cell Culture
Bovine aorta endothelial cells (BAECs) passage 5 to 7 were cultured in DMEM (Invitrogen) containing 4.5 mmol/L glucose supplemented with 10% FBS. Cells were treated in 100-mm dishes supplemented 70% confluence.
eNOS Activity
Enzyme activity was determined from BAECs stimulated with 1 µmol/L bradykinin for 30 minutes in Hank?s balanced saline solution (HBSS) containing 0.5 mmol/L L-arginine. The nitrite (NO 2 – ) accumulation was quantified by chemiluminescence using an NO Analyzer (Sievers Model 280i) and results were normalized to protein content.
2-Hydroxyethidium (2-OHE + ) Quantification in BAECs
The O 2 – production in BAECs was measured with hydroethidine by quantifying 2-OHE + by HPLC with electrochemical detection as described previously 25 (supplemental material, available online at http://atvb.ahajournals.org).
Tetrahydrobiopterin, Dihydrobiopterin, and Ascorbate Measurements
Quantification of BH 4, BH 2, and ascorbate was performed by HPLC method with electrochemical detection (EC-HPLC) as described in supplemental material.
GTP Cyclohydrolase-I Activity
After treatment with 4-HNE, cells were washed with DPBS and scraped into 50 mmol/L Tris-HCl buffer pH 7.4 containing 1 mmol/L magnesium chloride, 0.1 mol/L potassium chloride, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L PMSF, and protease inhibitor cocktail (Roche). Aliquots of 100 µL were assayed for activity as described previously. 17
Caspase Assay
Caspase-3 activity was measured as previously described using the fluorogenic substrate DEVD-AFC. 26
Proteasome Activity
Activity of the 26S proteasome was measured after the cleavage of the fluorogenic substrate Suc-LLVY-AMC (chymotrypsin-like) or Boc-Val-Leu-Lys-AMC (trypsin-like) as described in supplemental material.
GSH Assay
GSH levels were measured by the fluorescent HPLC-based analysis of 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBDF) derived products as described previously. 27
Statistical Analysis
Data are plotted as Mean±SD. Data Plotting and statistical analysis (ANOVA) were performed using Origin software (OriginLab). A probability value <0.05 was considered statistically significant from controls.
Results
4-HNE Stimulates O 2 – Production in Endothelial Cells
Incubations with 4-HNE were performed in serum-free media to avoid 4-HNE unspecific reactions via condensation with amino groups from proteins and free amino acids present in the complete media. Even so, free 4-HNE concentrations were decreased on diluting 4-HNE with culture media only ( Figure 1 ). This result was attributed to 4-HNE scavenging by essential amino acids such as histidine and lysine present in the culture media (DMEM, Invitrogen) in concentrations 0.7 and 0.2 mmol/L, respectively. In the presence of cells, 4-HNE disappearance from the media was increased. Under these conditions intracellular concentrations of 0.46±0.06 nmoles/mg protein ie, about 0.5 µmol/L were reached after 4-hour incubation with 25 µmol/L 4-HNE ( Figure 1 ). A decrease of about 30% was detected at longer incubation times, although 4-HNE uptake from the media was uninterrupted ( Figure 1 ). The 4-HNE Michael adducts with intracellular proteins shown to increase 4-hour incubations with 4-HNE concentrations 10 µmol/L (not shown). Thus the effects of 4-HNE on NO and superoxide production were examined at 4 hours, which allows 4-HNE to reach intracellular targets but short enough to reduce cellular stress caused by serum deprivation. 28
Figure 1. 4-HNE uptake by BAECs. A, Intracellular 4-HNE concentrations in BAECs treated with 4-HNE in serum free DMEM media. B, Extracellular 4-HNE concentrations on addition of 25 µmol/L 4-HNE in DMEM media with or without BAECs. Mean±SD. * P <0.05 vs time 0 controls (n=3).
After incubation with 4-HNE (0 to 25 µmol/L), eNOS activity in BAECs was stimulated with bradykinin and L-arginine in HBSS. Quantification of nitrite in the media showed that 4-HNE inhibits eNOS in a dose-dependent fashion ( Figure 2 ). To ensure this inhibition was not a direct effect on the enzyme, incubation of 4-HNE (0.2 to 0.05 mmol/L) with recombinant eNOS (1 µg) for 4 hours showed not to alter eNOS activity of 129.3±2.4 nmol citrulline/min/mg protein. Thus, eNOS inhibition in BAECs is most likely attributable to 4-HNE actions on other components of the eNOS system and not a direct effect on the protein itself.
Figure 2. 3). * P <0.05 vs untreated, nonsupplemented control. P <0.05 vs 4-HNE–treated, nonsupplemented control.
Because 4-HNE causes endothelial oxidative stress, 20–22 it was anticipated that this treatment would lead to a defective BH 4 supply resulting from changes in the redox state of BAECs. This mechanism was tested in cells supplemented with sepiapterin which provided eNOS with a significant protection against loss of activity ( Figure 2 A). This result suggested that loss of BH 4 is involved in 4-HNE mediated inhibition of eNOS activity. To better characterize this possibility, BAECs were treated with 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTPCH and BH 4 synthesis. This treatment decreased NO formation although to a lesser extent that 25 µmol/L 4-HNE. In addition, we tested the effect of ascorbate which is anticipated to protect cells from 4-HNE by ascorbylation 29 and shielding BH 4 from oxidation. 30 Intracellular ascorbate levels were increased over 400-fold (3.4±0.6 pmol/mg protein to 1.54±0.8 nmol/mg protein) by supplementing cells with 75 µmol/L ascorbate for 12 hours. As shown in the Figure 2 (panel B), at these concentrations ascorbate diminished eNOS inactivation indicating a protection. This protection however was less evident than that offered by sepiapterin ( Figure 2A and 2 B). The degree of NO inhibition by 4-HNE was comparable to the inhibition promoted by 0.1 mmol/L L-NAME as inferred from control experiments ( Figure 2 B).
Under the same experimental conditions, 4-HNE increased O 2 – production in BAECs in a dose-dependent manner as shown by the quantification of 2-hydroxyethidium by HPLC with electrochemical detection. Supplementation of the cells with ascorbate and sepiapterin decreased O 2 – detection. However, because sepiapterin decreased O 2 – and also increased NO formation, a mechanism involving eNOS uncoupling was postulated to contribute to increased O 2 – in response to 4-HNE challenge. Control experiments with L-NAME showed a partial protection of 4-HNE stimulated O 2 – increases ( Figure 2 D). This partial inhibition is in agreement with the modest effect of L-NAME on superoxide release from eNOS as previously shown by electron paramagnetic resonance spin trapping studies with purified eNOS. 31
Endothelial Oxidative Stress and Caspase Activity
Intracellular glutathione (GSH) levels were examined to assess the extent of oxidative stress induced by 4-HNE at the applied doses in BAECs. After 4 hours of treatment with 4-HNE, a moderate decrease in GSH was detected (supplemental Figure I). Ascorbate pretreatment did not significantly change this response. Increased caspase-3 activity, however, was stimulated at 4-HNE concentrations of about 20 µmol/L during the same period of time (supplemental Figure II). Although GSH levels likely reflect both consumption and rapid upregulation of biosynthesis, 20 this compensation did not prevent 4-HNE apoptosis signaling. However, sepiapterin supplementation significantly inhibited caspase-3 activation. This result suggested that the cytotoxic threshold of 4-HNE can be modulated by preventing eNOS uncoupling ie, increasing O 2 – in detriment of NO formation.
4-HNE Induces BH 4 Depletion via Inactivation of GTPCH
BH 4 levels were quantified by a new HPLC protocol with electrochemical detection. This analysis was less ambiguous and more sensitive (0.5 pmoles) than the fluorescent method based on differential acid-base KI/I 2 oxidation. Also the quantification of 7,8-dihydrobiopterin (BH 2 ) and ascorbate in the same sample injection is feasible ( Figure 3 A). In this analysis, it was shown that 4-HNE depleted BH 4 to the same extent than treatment with the GTPCH inhibitor DAHP ( Figure 3 B). The BH 4 depletion, however, was not followed by an increase in BH 2 levels. Supplementation of cells with sepiapterin increased BH 4 content several-fold, and remained marginally affected by 4-HNE. Ascorbate increased basal levels of BH 4, but this increase was not sufficient to counteract BH 4 depletion following 4-HNE treatment ( Figure 3 C). In combination these results indicated that 4-HNE depleted BH 4 by mechanisms other than direct BH 4 oxidation to BH 2, because this product was not detected, and ascorbate offered only a partial protection. Activity measurement of GTP cyclohydrolase I (GTPCH), the first enzyme in the BH 4 biosynthetic pathway, showed GTPCH inactivation on 4-HNE treatment. Together these results indicated that 4-HNE decreases BH 4 availability by targeting its de novo biosynthesis. Covalent modification of proteins by 4-HNE has been linked to loss of functions and increased proteasomal degradation. To examine this possibility, BAECs were pretreated with lactacystin to examine the effects of proteasome inhibition in the loss of BH 4. Unlike ascorbate, lactacystin treatment did not increase basal BH 4 levels, however it did protect cells from 4-HNE induced BH 4 loss suggesting that 4-HNE via proteasome activation decreases BH 4 levels in BAECs.
Figure 3. 4-HNE decreases BH 4 levels and GTPCH activity. A, EC-HPLC elution profile of 25 pmoles BH 4, BH 2 3). * P <0.05 vs untreated, nonsupplemented control. P <0.05 vs 4-HNE–reated, non-treated supplemented control.
4-HNE Downregulates GTPCH and hsp90 Protein Levels but not eNOS
Treatments with 4-HNE did not alter eNOS, whereas a significant downregulation of hsp90 and GTPCH protein levels were induced by 4-HNE (25 µmol/L; Figure 4 ). The loss of hsp90 and GTPCH protein was offset by pretreatment with sepiapterin ( Figure 4 ). Control experiments with the protein-synthesis inhibitor cycloheximide (10 µmol/L, 4 hours) indicated that 4-HNE does not interfere with the synthesis of new protein (data not shown). Thus, the reduced levels of GTPCH and hsp90 are most likely attributable to stimulated protein degradation. Lactacystin, a powerful inhibitor of the ubiquitin-proteasome system, protected hsp90 and GTPCH from 4-HNE–mediated downregulation.
Figure 4. 3). * P <0.05 vs control.
4-HNE Increased Protein Ubiquitinylation and 26S Proteasomal Activity
The involvement of the ubiquitin-proteasome pathway in the effects of 4-HNE in BAECs was further indicated by the increase in polyubiquitinylated proteins ( Figure 5 ). Pretreatment of BAECs with MG132, an unspecific proteasome and lysosomal inhibitor, did not change the accumulation of ubiquitinylated protein whereas a moderate but significant increase was seen on lactacystin pretreatment. Also 4-HNE stimulated the 26S chymotrypsin activity of the proteasome as shown in the activity assays ( Figure 5 B).
Figure 5. 4-HNE increases protein ubiquitinylation and 26S proteasome activity. A, BAECs were pretreated for 24 hours with 10 µmol/L MG132 or lactacystin followed by 4-HNE. Cell lysates were analyzed by Western blotting. B, Lactacystin inhibitable 26S-proteasome activity. Data are shown as Mean±SD (n=3). * P <0.05 vs control.
4-HNE Decreases eNOS-Serine 1179 Phosphorylation
Diminished eNOS-serine 1179 levels were shown on 4-HNE incubations ( Figure 6 ). Thus an additional mechanism by which 4-HNE disarranges NO production is occurring through disruption of eNOS-phosphorylation. Furthermore, treatment with 4-HNE did not change the eNOS-dimer abundance at any of the tested concentrations ( Figure 6 ). These results are in agreement with controls showing that eNOS in untreated cells is found in a dimeric state that is not changed after BH 4 depletion with the inhibitors DAHP+N-acetyl serotonin or supplementation with sepiapterin (supplemental Figure III). These data further indicate that 4-HNE actions on eNOS activity are not attributable to modification of eNOS residues that could lead to dissociation of eNOS into inactive monomers in cells.
Figure 6. eNOS-serine 1179 and eNOS monomer:dimer in 4-HNE treated cells. A, Proteins were analyzed by Western blotting. B, eNOS dimer:monomer in freshly lysed cells. Denaturating gels contained β-mercaptoethanol. Samples were boiled (95°C) or not (4°C) for 10 minutes. Gels were maintained at 4°C and analyzed by Western blot.
Discussion
It has been anticipated that lipid peroxidation products play a role in the mechanisms mediating vascular injury. Bifunctional electrophiles such as 4-HNE have been shown to signal several processes in endothelial cells including activation of signaling pathways via extracellular signal regulated kinase (ERK), JNK, and p38MAPK, modification of cell adhesion properties, and mediating protein inactivation via modification of lysine, histidine, and cysteine residues. The endothelial responses to electrophilic lipids, however, has been shown to be dependent on concentrations. Whereas high concentrations of 4-HNE are deemed cytotoxic, lower concentrations have been shown cytoprotective via transcriptional regulation of antioxidant genes affecting GSH and heme oxygenase levels. 32 The transcriptional induction of GSH synthesis by 4-HNE is linked to activation of the antioxidant response elements (AREs) via modification of critical thiols in Keap-1 ( K elch-like erythroid cell-derived protein with CNC homology ( E CH)- a ssociated p rotein 1), 32 a known modulator of ARE.
The cellular responses to 4-HNE are important in determining its role in the progress of disease. The exact events involved in the shift of cellular responses to 4-HNE however have been more challenging to establish. This is in part linked to the high variability of experimental conditions that ultimately determine 4-HNE availability to endothelial cells. Here we show that uptake of free 4-HNE by BAECs is slower than previously assumed. The accumulation of 4-HNE in BAECs did not deplete GSH levels to the extent anticipated if GSH were the main target of 4-HNE reactions. A possible compensation via the activation of AREs may be possible, but in the time frame of our experiments this response needs to be determined.
The direct involvement of lipid peroxidation products in increasing reactive oxygen species formation has been suggested but has been difficult to demonstrate. Here we report that 4-HNE inhibits endothelial NO generation by a mechanism involving BH 4 depletion and inhibition of eNOS-S1179 phosphorylation. These changes alone could explain 4-HNE increased oxidative stress. However we also show that these responses are followed by increased O 2 – which will exacerbate oxidant stress induced by 4-HNE. Sepiapterin supplementation counteracted the loss of NO and accumulation of O 2 – indicating that 4-HNE by targeting the BH 4 pathway mediates eNOS uncoupling. Previous studies have shown that H 2 O 2 modulates eNOS expression and phosphorylation depending on their concentrations in endothelial cells. 33 Whereas low H 2 O 2 levels increase NO formation via stimulated eNOS phosphorylation, high levels have opposite effects. Oxidized LDL, possibly via 4-HNE, also has been shown to increase eNOS phosphorylation in endothelial cells 34 that are in agreement with a mechanism producing low levels of ROS. If levels of ROS are the most important variable modulating cytotoxicity, thus it is possible to conclude that eNOS uncoupling further increases ROS production to cytotoxic levels.
Regulation of BH 4 concentrations in endothelial cells is not fully understood, however continuous synthesis via GTPCH is absolutely required to ensure even low basal concentrations. 35 We have shown that 4-HNE diminishes BH 4 by a mechanism that does not involve oxidation to BH 2, but GTPCH degradation via proteasome-inhibitable pathway. Sepiapterin and ascorbate supplementation counteracted loss of BH 4 and eNOS uncoupling ie, loss of NO and increase O 2 – production. It is likely that ascorbate acts by direct scavenging of 4-HNE 29 as high concentrations of ascorbate were necessary to reach an effect. Sepiapterin however by readily increasing BH 4 levels and preventing eNOS uncoupling was very efficient at inhibiting both O 2 – with concomitant increase in NO formation.
There is evidence that endothelial proteasome regulates eNOS activity. 36 Also it has been shown that basal NO by regulating the immunoproteasome and proteasomal activity protects cells from apoptosis. 37 Although 4-HNE did not directly affect eNOS properties, it mediates a process involving protein ubiquitinylation and proteasome degradation of GTPCH. This leads to a condition of chronic NO deficiency and oxidative stress as eNOS becomes uncoupled. Restitution of NO production by sepiapterin supplementation decreased loss of GTPCH, indicating the role of NO in proteasome activation. Hsp90 is also a target of proteasome activity, which aggravates the loss of NO production as indicated by the diminished levels of phosphorylated eNOS-serine 1179. Covalent modification of hsp90 on the cys-572 by 4-HNE has been associated with its loss of chaperone activity in a model of chronic alcoholic liver disease. 38 Whether this modification also explains the loss of hsp90 and impaired eNOS-serine 1179 phosphorylation in 4-HNE–treated cells remains to be demonstrated.
In conclusion, the present study indicates that 4-HNE mediates endothelial oxidative stress through a series of complex processes involving loss of BH 4 and NO and alterations in the proteasome activity. Recently it was shown that electrophilic lipids such as 15-A 2t -isoprostane and 4-HNE localize in the mitochondria increasing O 2 – release. 39 Although this phenomenon has been attributed to increased formation of protein adducts, it is conceivable that inhibition of NO formation also plays a role. Diminished NO levels will increase oxygen reduction at the cytochrome c oxidase level in the mitochondrial respiratory chain. 40 While this reaction is anticipated to decrease O 2 – production in the mitochondria, the simultaneous decrease in reaction of O 2 – with NO may balance the effect toward increased O 2 – formation. As NO influences the spectrum of oxidants generated in the mitochondria, it may also influence compensatory mitochondrial responses such as mitochondrial biogenesis as proposed. 41 Thus, it is anticipated that inhibition of NO by 4-HNE has a major impact in the effects on mitochondria dysfunction and oxidant generation in the endothelium. As shown in the supplemental Scheme I, tetrahydrobiopterin supplementation may exert a protective role in different states of disease where ROS production becomes a prominent event. But also preventing eNOS uncoupling may be important in disrupting a feedback mechanism involving increasing ROS and lipid peroxidation that may well explain the events leading to loss of endothelial function, increased oxidative stress, and accumulation of electrophilic lipids in atherosclerotic lesions. Thus in establishing a role of 4-HNE as a redox signaling molecule in the endothelium the impact of eNOS uncoupling to increase ROS formation needs to be considered. This may shed light on current controversy regarding the role of electrophilic lipids in the regulation of ROS production in the endothelium. 42
Acknowledgments
We are thankful for the support of the Free Radical Research Center at the Medical College of Wisconsin.
Sources of Funding
This work was funded by the National Institutes of Health award HL67244 and P20 RR17699-01 COBRE.
Disclosures
None.
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作者单位：Department of Biophysics (J.W.) and Free Radical Research Center (J.V.V.), Medical College of Wisconsin, Milwaukee; and the Department of Pharmacology, Physiology, and Therapeutics (M.J.P.), University of North Dakota, Grand Forks.