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Am J Physiol Cell Physiol 293: C1679-C1686, 2007. First published September 19, 2007; doi:10.1152/ajpcell.00224.2007
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VASCULAR BIOLOGY

Role of DDAH-1 in lipid peroxidation product-mediated inhibition of endothelial NO generation

Arthur J. Pope,1 Lawrence Druhan,2 Jorge E. Guzman,2 Scott P. Forbes,1 Velayutham Murugesan,2 Deqin Lu,2 Yong Xia,2 Louis G. Chicoine,3 Narasimham L. Parinandi,2 and Arturo J. Cardounel1

Davis Heart and Lung Research Institute, Departments of 1Pharmacology and 2Internal Medicine, The Ohio State University College of Medicine; and 3Columbus Children's Research Institute, Columbus, Ohio

Submitted 29 March 2007 ; accepted in final form 10 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Altered nitric oxide (NO) biosynthesis is thought to play a role in the initiation and progression of atherosclerosis and may contribute to increased risk seen in other cardiovascular diseases. It is hypothesized that altered NO bioavailability may result from an increase in endogenous NO synthase (NOS) inhibitors, asymmetric dimethly araginine (ADMA), and NG-monomethyl arginine, which are normally metabolized by dimethyarginine dimethylamine hydrolase (DDAH). Lipid hydroperoxides and their degradation products are generated during inflammation and oxidative stress and have been implicated in the pathogenesis of cardiovascular disorders. Here, we show that the lipid hydroperoxide degradation product 4-hydroxy-2-nonenal (4-HNE) causes a dose-dependent decrease in NO generation from bovine aortic endothelial cells, accompanied by a decrease in DDAH enzyme activity. The inhibitory effects of 4-HNE (50 µM) on endothelial NO production were partially reversed with L-Arg supplementation (1 mM). Overexpression of human DDAH-1 along with antioxidant supplementation completely restored endothelial NO production following exposure to 4-HNE (50 µM). These results demonstrate a critical role for the endogenous methylarginines in the pathogenesis of endothelial dysfunction. Because lipid hydroperoxides and their degradation products are known to be involved in atherosclerosis, modulation of DDAH and methylarginines may serve as a novel therapeutic target in the treatment of cardiovascular disorders associated with oxidative stress.

nitric oxide; endothelial nitric oxide synthase; asymmetric dimethly araginine; 4-hydroxy-2-nonenal; dimethyarginine dimethylamine hydrolase

ENDOTHELIUM-DERIVED NITRIC OXIDE (NO) is a potent vasodilator that plays a critical role in maintaining vascular homeostasis through its anti-atherogenic and anti-thrombotic effects on the vascular wall (15, 29, 31). In this regard, impaired endotheliumderived NO production has been implicated in the pathogenesis of atheroproliferative disorders (10). Among the proposed mechanisms for the impaired NO synthase (NOS) activity observed in these conditions are the elevated levels of oxidatively modified lipids (18, 44). Polyunsaturated fats in cholesterol esters, phospholipids, and triglycerides are subjected to free radical-initiated oxidation. These polyunsaturated fatty acid peroxides can yield a variety of highly reactive smaller molecules such as the aldehyde 4-hydroxy-2-nonenal (4-HNE) upon further oxidative degradation (17). 4-HNE is a major biologically active aldehyde formed during lipid peroxidation of {omega}6 polyunsaturated fatty acids, which has been shown to accumulate in membranes at concentrations from 10 µm to 5 mM (40). There is a body of evidence that suggests that reactive aldehydes such as 4-HNE play a role in the progression of atherosclerosis. Plasma concentrations of these reactive aldehydes are known to increase relative to the progression of aortic atherosclerosis, and during the oxidation of LDL, high concentrations of the reactive aldehydes are generated (9, 30). It has been suggested that the elevations in these highly reactive lipid hydroperoxide degradation products result in impaired endothelial function and atherosusceptibility, secondary to NOS impairment (32, 35, 44). In support of the importance of the reactive aldehyde involvement in endothelial dysfunction, here we demonstrate that exposure of aortic endothelial cells to 4-HNE dose dependently inhibits NO bioavailability. We hypothesize that the decrease in NO bioavailability is a result of increased levels of the NOS inhibitors asymmetric dimethly arginine (ADMA) and NG-monomethyl arginine (NMMA).

ADMA has been shown to be increased in conditions associated with increased risk of atherosclerosis and independently predicts total and cardiovascular mortality in individuals with angiographic coronary artery disease (2, 27, 28, 33, 36, 38). However, little is known with regard to the pathways leading to the methylarginine accumulation observed in cardiovascular diseases. These endogenous inhibitors of NOS are derived from the proteolysis of methylated arginine residues in various proteins. The methylation is carried out by a group of enzymes referred to as protein-arginine methyl transferases. Subsequent proteolysis of proteins containing methylarginine groups leads to the release of free methylarginine into the cytoplasm where NO production from NOS is inhibited (4, 11, 23). These methylarginines are subsequently degraded by the enzyme dimethylarginine dimethylamine hydrolase (DDAH), which hydrolyzes the conversion of ADMA to L-citrulline and dimethylamine (19, 25). The activity of DDAH has been shown to be decreased by oxidized LDL and tumor necrosis factor-{alpha}, yielding increased methylarginine levels with subsequent impairment of NOS-derived NO generation (8, 12, 16, 24). Because NO is known to possess anti-proliferative and anti-atherogenic properties, methylarginine accumulation in response to the decreased DDAH expression/activity has been proposed to be involved in the vascular pathophysiology observed in a variety of cardiovascular diseases (3, 5, 23, 41, 42). However, the mechanisms as to how methylarginines are modulated and what role they play in disease progression are not understood. Therefore, the current studies were performed to establish the effects of the lipid peroxidation degradation product 4-HNE on NO production and determine whether methylarginines are involved in the lipid peroxidation-mediated pathogenesis of endothelial dysfunction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. 4-HNE was purchased from Biomol (Plymouth Meeting, PA). BAECs were purchased from Cell-Systems (Kirkland, WA). All other reagents were purchased from Sigma (St. Louis, MO).

Cell culture. Bovine aortic endothelial cells (BAECs) were purchased from Cell Systems and cultured in DMEM containing 10% fetal bovine serum, 1% nonessential amino acid solution, 0.2% endothelial cell growth factor supplement, and 1% antibotic-antimyotic and incubated at 37°C under a humidified environment containing 5% CO2-95% O2. For experiments involving exposure to 4-HNE, 4-HNE was prepared as a stock solution in ethanol at a concentration of 50 mM. The 4-HNE was then added to the media of BAECs and incubated for 24 h. Dilutions of 4-HNE were performed to maintain the final ethanol concentration below 0.2%.

Electron paramagnetic resonance spectroscopy and spin trapping. Spin-trapping measurements of NO were performed using a Bruker EPR 300 spectrometer with Fe-N-methyl-D-glucamine dithiocarbamate (Fe-MGD) as the spin trap (5, 7). For measurements of NO produced by BAECs, cells were cultured as described above, and spin-trapping experiments were performed on cells grown in six-well plates (1 x 106 cells/well). In these studies, cells attached to the substratum were utilized since scraping or enzymatic removal leads to injury and membrane damage with impaired NO generation. The medium from each well was removed, and the cells were washed three times with phosphate-buffered saline (without CaCl2 or MgCl2). Next, 0.3 ml of PBS containing glucose (1 g/l), CaCl2, MgCl2, the NO spin-trap FE-MGD (0.5 mM Fe2+, 5.0 mM MGD), and calcium ionophore (1 µM) was added to each well, and the plates were incubated at 37°C under a humidified environment containing 5% CO2-95% O2 for 30 min. After incubation, the medium from each well was removed, and the trapped NO in the supernatants was quantified using electron paramagnetic resonance. Spectra recorded from these cellular preparations were obtained using the following parameters: microwave power, 20 mW; modulation amplitude, 3.16 G; and modulation frequency, 100 kHZ.

Measurement of endothelial cell ADMA and L-Arg levels. BAECs were collected from confluent 75-cm2 culture flask by gentle scraping followed by sonication in PBS followed by extraction using a cation-exchange column. Samples were derivatized with OPA and separated on a Supelco LC-DABS column (4.6 mm x 25 cm ID, 5 µm particle size), and L-Arg and methylarginines were separated and detected using an ESA (Chelmsford, MA) HPLC system with electrochemical detection at 400 mV (7). Intracellular levels of L-Arg and methylarginines were determined from values derived from standard curves of each analyte using the ESA peak integration software assuming intracellular water content of 2 pL.

DDAH-1 and eNOS expression. DDAH-1 was detected by anti-DDAH-1 goat IG purchased from IMGENEX and diluted 1:2,000 (San Diego, CA). eNOS was detected using an anti-eNOS antibody purchased from Calbiochem (San Diego, CA). The secondary antibodies were donkey anti-goat IgG-horseradish peroxidase and goat anti-rabbit IgG-horseradish peroxidas, respectively, and purchased from Santa Cruz (Santa Cruz, CA). The secondary antibodies were diluted 1:2,000. Western blot detection was performed using an enhanced chemiliumnesece kit purchased from Amersham Biosciences (Piscataway, NJ).

DDAH activity. DDAH activity was measured from the conversion of L-[14C]NMMA to L-[14C]citrulline. BAECs grown to confluence in T-75 flasks were trypsinized, pelleted, and resuspended in 150 µl of 50 mM Tris (pH 7.4). The cells were then sonicated 4 x 2 s, and 150 µl of the reaction buffer (50 mM Tris, 20 µM L-[14C]NMMA, 180 µM L-NMMA, pH 7.4) were added to each sample. The samples were then incubated in a water bath at 37°C for 90 min. After the incubation, the reaction was stopped with 1 ml of ice-cold stop buffer using 20 mM HEPES with 2 mM EDTA, pH 5.5. Separation of L-[14C]citrulline from L-[14C]NMMA was performed using the cation exchange resin Dowex AG50WX-8 (0.5 ml, Na+ form, Pharmacia). The L-[14C]citrulline in the eluent was then determined using a liquid scintillation counter.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of 4-HNE on endothelial cell NO production. Previous studies have demonstrated that lipid hydroperoxide levels are elevated in atherosclerotic lesions, and the presence of these oxidized lipid congeners may contribute to the endothelial dysfunction observed in CAD (9, 30). Therefore, to determine the effects of lipid hydroperoxides on endothelial function, cellular studies were carried out using BAECs stimulated with calcium ionophore to assess the effects of 4-HNE on NO production from endothelial cells. Endothelial cells were cultured in six-well plates and upon reaching confluence were exposed to 4-HNE (10–100 µM) for 24 h. 4-HNE was dissolved in ethanol and added directly to the media (0.1% EtOH). This compound is highly lipophilic and readily crosses cellular membranes, as such no carrier is needed to deliver this agent into the cell (13). At the end of the incubation period, EPR spin-trapping measurements were performed to measure endothelium-derived NO production. Results demonstrated that 4-HNE dose dependently inhibited NO generation from BAECs, with 10 µM 4-HNE inhibiting NO generation by 14%, 50 µM inhibited NO generation by 45%, and at 100 µM a 72% inhibition was observed (Fig. 1). Results from these studies demonstrated that 4-HNE, at pathologically relevant levels, dose dependently inhibited eNOS-derived NO generation. Measurements of cell viability demonstrated no increase in cell death with 4-HNE doses up to 50 µM; however, at 100 µM 4-HNE cell viability decreased by 18%. Thus all subsequent studies were performed using 4-HNE concentrations ≤50 µM. Control studies using the nonoxidized carbonyl hexanol demonstrated no significant inhibition in cellular NO production with concentrations up to 50 µM. The concentration of 4-HNE (50 µM) used for subsequent studies represents pathologically relevant levels of this reactive lipid oxidation product as previous studies have shown concentrations exceeding 50 µM 4-HNE in the plasma of dogs following reperfusion injury (26, 34).


Figure 1
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  		Fig. 1. Effects of 4-hydroxy-2-nonenal (4-HNE) on nitric oxide (NO) production. NO generation from bovine aortic endothelial cells (BAECs) stimulated with calcium ionophore A23187(1 µM) was measured by electron proteon resonance (EPR) spin trapping with the Fe-N-methyl-Dglucamine dithiocarbamate (Fe-MGD) complex. Left: amplitude of the NO triplicate EPR spectrum over 10 consecutive 1-min scans after a 30-min incubation period. Right: EPR spectra and the dose-dependent effects of the 4-HNE treatment on NO production. Results are means ± SD. *Significance at P < 0.05.

 
Effect of 4-HNE on eNOS expression. To determine whether the inhibitory effects of 4-HNE on cellular NO production were due to alterations in eNOS expression, Western blot analysis was performed and measurements of eNOS expression were carried out (Fig. 2A). NOS phosphorylation status was measured using the Western blot techniques with antipSer1179 and pThr-497 antibodies (Fig. 2A). In addition, because calcium ionophore stimulation is know to alter Ser1179 phosphorylation, Western blot experiments were also performed on A23187 [GenBank] -stimulated BAECs following 4-HNE treatment (Fig. 2B). Results demonstrated that the loss of NOS activity was independent of both eNOS protein expression and phosphorylation status as both of these outcomes were unchanged following exposure to 4-HNE.


Figure 2
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  		Fig. 2. 4-HNE effects on endothlial NO synthase (eNOS) expression and phosphorylation. Protein was obtained from BAECs treated with varying concentrations (1–50 µM) of 4-HNE for 24 h. A: for measurements of eNOS, Ser 1179-phosphorylated eNOS (p-eNOSser1179), and Thr497-phosphorylated eNOS (p-eNOSthr497) expression, 30 µg of protein was loaded into each well. Lane 1 is the ladder, lane 2 is nontreated cells, and lanes 3–6 are cells treated with 4-HNE. B: effects of 4-HNE on Ser1179 phophorylation following calcium ionphore (5 µM, A23187) stimulation.

 
Restoring NO generation from cells. Because the observed NO inhibition did not result from changes in protein expression or phosphorylation state, we carried out additional studies aimed at assessing whether substrate or cofactor depletion was involved in the observed decrease in NO bioavailability. In this regard, oxidant stress, which has been shown to occur following exposure to lipid oxidation products, has been shown to reduce the bioavailability of the critical NOS cofactor BH4 (21, 40, 43). Loss of this cofactor results in NOS uncoupling with the enzyme primarily generating superoxide. Moreover, oxidant injury has also been demonstrated to increase cellular levels of the endogenous methylarginine ADMA (22). Therefore, cellular studies were carried out to investigate the effects of adding both an antioxidant to prevent BH4 oxidation as well as the eNOS substrate L-Arg, to overcome endogenous methylarginine-mediated NOS inhibition. Results demonstrated that 24 h exposure of BAECs to 50 µM 4-HNE resulted in a 51% decrease in endothelial NO generation (Fig. 3). When these experiments were repeated in the presence of GSH (1 mM) or with L-Arg supplementation (1 mM), NO production increased by 26% and 7%, respectively. Moreover, when these experiments were repeated in the presence of both GSH and L-Arg, endothelial cell NO production was restored to near-normal levels (87% of control) (Fig. 3). These results suggest that 4-HNE-mediated effects on NO production involve multiple mechanisms, which include elevated levels of methylarginines.


Figure 3
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  		Fig. 3. Effects of L-Arg and GSH supplementation on NO generation. NO generation from BAECs stimulated with calcium ionophore A23187 (1 µM) was measured by EPR spin trapping with the Fe-MGD complex. Experimental groups consisted of untreated (Control), 1 mM L-Arg supplementation, 1 mM GSH supplementation (GSH), and 1 mM L-Arg with 1 mM GSH (L-Arg + GSH). These experiments were performed in both the presence and absence of 4-HNE (50 µM). Results are means ± SD. *Significance at P < 0.05.

 
Effects of 4-HNE on cellular ADMA levels. Our observation that 4-HNE treatment impairs cellular NO production and that this inhibitory effect can be reversed with L-Arg administration suggests that intracellular levels of the NOS inhibitor ADMA may be elevated. To confirm this hypothesis, cellular levels of ADMA and L-Arg were measured following exposure of BAECs to 50 µM 4-HNE for 24 h. Results demonstrated that at 24 h postexposure to 4-HNE, endothelial cell concentrations of ADMA increased from 3.2 ± 0.5 to 6.5 ± 0.7 µM, whereas L-Arg levels were not significantly different (Fig. 4). These results support our conclusion that the inhibitory effects of 4-HNE on endothelial NO production are due, at least in part, to the increased levels of the competitive NOS inhibitor ADMA.


Figure 4
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  		Fig. 4. Effects on 4-HNE on the levels of asymmetric dimethly arginine (ADMA) in BAECs. BAECs were cultured in T75 flasks and exposed to 50 µM 4-HNE for 24 h. Total cellular ADMA levels were measured using HPLC techniques. Results are means ± SD. *Significance at P < 0.05.

 
Effect of 4-HNE on DDAH expression and activity. Cellular methylarginine levels are regulated by DDAH, the enzyme responsible for the metabolism of both ADMA and L-NMMA. Recent studies have demonstrated that the expression and activity of this methylarginine-regulating enzyme decreases in variety of cardiovascular diseases. Therefore, to determine whether the observed elevations in intracellular ADMA were a result of changes in DDAH, measurements of DDAH expression and activity were performed following exposure of BAECs to 4-HNE. BAECs were treated with 4-HNE (50 µM) followed by Western blot analysis and enzyme activity assays. Results demonstrated that exposure of endothelial cells to 4-HNE did not affect the protein expression (Fig. 5A) but resulted in a 40% decrease in cellular DDAH activity (Fig. 5B). Studies were then performed with purified recombinant human (h)DDAH-1 to evaluate whether the observed cellular inhibition of DDAH activity was a result of direct 4-HNE effects on the enzyme. Incubation of purified hDDAH-1 with 50 µM 4-HNE resulted in a 41% decrease in activity from the purified enzyme (Fig. 5B). This loss in activity was largely restored by GSH (1 mM) preincubation. Together, these results demonstrate that 4-HNE directly inhibits DDAH activity resulting in increased methylarginine levels and thus impaired eNOS-derived NO.


Figure 5
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  		Fig. 5. 4-HNE effects on dimethyarginine dimethylamine hydrolase (DDAH) expression and activity. A: DDAH-1 expression was measured by Western blot techniques from BAECs treated with 50 µM 4-HNE for 24 h. B: DDAH activity was measured from BAEC homogenates following a 24-h incubation with 4-HNE (50 µM). DDAH activity was measured from purified recombinant hDDAH-1 (5 µg) following a 60-min incubation with 50 µM 4-HNE. Experimental groups consisted of 50 µM 4-HNE and 50 µM 4-HNE + 1 mM GSH. Results are means ± SD. *Significance at P < 0.05.

 
Effects of DDAH overexpression on endothelial NO production following exposure to 4-HNE. Our results have demonstrated that the exposure of BAECs to the lipid peroxidation product 4-HNE results in the impaired NO production and accumulation of ADMA, secondary to the loss of DDAH activity. Therefore, studies were carried out to determine whether overexpression of DDAH-1 could restore endothelial NO production following 4-HNE challenge. BAECs were grown to 80% confluence and then transduced with adDDAH-1 [25 multiplicities of infection (MOI)], which resulted in a threefold increase in DDAH 1 expression. After 24 h of adenoviral transfection, cells were challenged with 4-HNE and allowed to incubate for an additional 24 h. At the end of the 24-h challenge, EPR analysis of NO production was carried out as described in the MATERIALS AND METHODS. Results demonstrated that exposure to 4-HNE (50 µM) resulted in a 36% decrease in NO generation in cells transduced with a control vector (Fig. 6). Cells overexpressing DDAH-1 demonstrated a 22% basal increase in NO generation compared with the control vector, suggesting that the endogenous levels of methylarginine are sufficient to significantly inhibit cellular NO production (Fig. 6). Exposure of cells overexpressing DDAH-1 to 4-HNE (50 µM) resulted in a 58% decrease in NO production, thus demonstrating that DDAH alone cannot restore eNOS function. However, when these experiments were repeated in the presence of GSH, DDAH overexpression was able to almost completely restore NO production following 4-HNE challenge, whereas GSH alone had only modest effect (Fig. 6).


Figure 6
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  		Fig. 6. Effects of DDAH overexpression on endothelial cell NO production following 4-HNE challenge. NO generation BAECs stimulated with calcium ionophore A23187 (1 µM) was measured by EPR spin trapping with the Fe-MGD complex. Experimental groups consisted control vector (Control), DDAH overexpressing (DDAH), glutathione (1 mM) treated (GSH), and DDAH overexpression with GSH (1 mM) treatment (DDAH + GSH). These experiments were performed in both the presence and absence of 4-HNE (50 µM). Results are means ± SD. *Significance at P < 0.05.

 
To confirm that these NO-restoring effects were dependent on increased DDAH activity, studies were performed measuring the conversion of L-[14C]NMMA to L-[14C]citrulline in BAECs. We found that exposure of BAECs to 4-HNE (50 µM) resulted in a 38% decrease in DDAH activity, supporting our previous HPLC results (Fig. 7). Overexpression of DDAH-1 increased DDAH activity by ~50%, and this increase in activity was reduced by 25% following exposure of BAECs to 4-HNE. Although DDAH activity was significantly higher in the DDAH-overexpressing cells exposed to 4-HNE compared with the control, this increase in DDAH activity was not accompanied by an in increased NO production (Fig. 6). Treatment of BAECs with the antioxidant GSH had modest effect on the DDAH activity and did not significantly prevent the loss of DDAH activity following 4-HNE challenge, whereas the combination of DDAH overexpression and GSH increased DDAH activity by ~50% (Fig. 7) with near-complete restoration of NO production (Fig. 6). These results suggest that 4-HNE causes NOS impairment through multiple mechanisms involving methylarginine accumulation and possibly NOS uncoupling. Evidence for NOS uncoupling is supported by our data demonstrating that DDAH overexpression in the presence of 4-HNE actually exacerbates the effects of 4-HNE on NO production (Fig. 6). In this regard, we have previously reported that methylarginines inhibit NOS-derived superoxide, and as such, overexpression of DDAH would reduce this inhibitory effect resulting in increased NOS-derived superoxide and reduced NO bioavailability (6). Evidence for the multiple mechanisms through which 4-HNE mediates its effects are supported by our results demonstrating that GSH treatment alone or DDAH overexpression alone has only moderate protection from 4-HNE-induced NOS dysfunction. However, in combination, these two treatments largely restored endothelial NO generation (Fig. 6). Therefore, complete protection of endothelium-derived NO generation from 4-HNE damage can only be achieved by both preventing NOS uncoupling (GSH treatment) and methylarginine accumulation (DDAH overexpression).


Figure 7
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  		Fig. 7. Effects of 4-HNE on endothelial cell DDAH activity. DDAH activity was assessed by measuring the conversion of L-[C14]NMMA to [C14]citrulline. Experimental groups consisted control vector (Control), DDAH overexpressing (DDAH), 1 mM glutathione supplementation (GSH), and DDAH overexpression with 1 mM GSH treatment (DDAH + GSH). These experiments were performed in both the presence and absence of 4-HNE (50 µM). Results are means ± SD. *Significance at P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
There is a growing volume of literature implicating ADMA as a key player in endothelial dysfunction and strong correlative data suggesting that ADMA is involved in the pathophysiology of a variety of cardiovascular diseases including hypertension and atherosclerosis (3, 23). More recently we and others (5, 20) have shown that methylarginines are elevated in response to vascular injury and that this elevation in ADMA and L-NMMA results in impaired endothelial function. In addition to mechanical injury, studies have also demonstrated that exposure of endothelial cells to pro-atherogenic lipoproteins such as LDL, results in increased cellular ADMA levels (16). Polyunsaturated fats in cholesterol esters, phospholipids, and triglycerides are subjected to free radical oxidation. These polyunsaturated fatty acids can yield a variety of lipid hydroperoxides and highly reactive lipid peroxidation products such as the aldehyde 4-HNE. During inflammation and oxidative stress, levels of 4-HNE have been shown to accumulate in membranes at concentrations from 10 µm to 5 mM (40). Moreover, studies have suggested that reactive aldehydes/carbonyls such as 4-HNE may play a critical role in the progression of atherosclerosis (9, 30). Plasma concentrations of these lipid peroxidation products are known to increase relative to the progression of atherosclerosis, and during the oxidation of LDL high concentrations of these reactive aldehydes/carbonyls are formed. We thus hypothesized that elevations in lipid peroxidation products may result in impaired endothelial function and atherosusceptibility, secondary to NOS impairment.

Therefore, studies were performed to determine the effects of the highly reactive lipid peroxidation product 4-HNE on endothelium-derived NO generation. Results demonstrated that the exposure of BAECs to 4-HNE caused a dose-dependent inhibition of cellular NO production. The observed 4-HNE effects were independent of changes in either NOS expression or phosphorylation state, because the Western blot analysis revealed no changes in either endpoint. These results suggested that the observed NOS impairment involved mechanisms other than those related to protein expression. As such, subsequent experiments were performed to determine whether alterations in NOS cofactors or substrate may be involved in the decreased NO bioavailability. In this regard, oxidant stress, which has been shown to occur following exposure to lipid peroxidation products, has been shown to reduce the bioavailability of the critical NOS cofactor BH4 (21, 40). Loss of this cofactor results in NOS uncoupling evident by impaired NO synthesis and enhanced superoxide production from the enzyme (43). Moreover, oxidant injury has also been demonstrated to increase the cellular levels of the endogenous methylarginine ADMA (23). Therefore, cellular studies were carried out to investigate the effects of adding both an antioxidant (GSH) to prevent BH4 oxidation as well as the eNOS substrate L-Arg to overcome endogenous methylarginine-mediated NOS inhibition. Our data demonstrate that the addition of either GSH or L-Arg alone had only modest NO-enhancing effects; however, coincubation with both GSH and L-Arg was able to almost completely restore endothelial NO production. These data suggest that the observed NOS impairment involves both oxidant-induced NOS inhibition (alleviated by the addition of GSH) as well as methylarginine accumulation (alleviated by the addition of excess substrate).

Direct measurement of ADMA levels and DDAH activity within cells by HPLC demonstrated that following 4-HNE challenge, intracellular ADMA levels were increased greater than twofold. Based on previously published studies demonstrating the kinetics of ADMA-mediated cellular inhibition, a twofold increase in methylarginine levels would be expected to inhibit NOS-dependent NO generation by 20–30% (5). The additional inhibition observed could be due to compartmentalization or NOS uncoupling and increased NOS-derived superoxide production in the presence of ADMA. To test this hypothesis, we used luminescence and Western blot studies to measure ONOO– levels and nitrotyrosine formation, respectively. Although no significant increase in ONOO– formation was observed, this does not rule out NOS uncoupling as superoxide generation from the enzyme is likely below detection limits. In this regard, we have also employed EPR spin-trapping techniques to measure eNOS-derived endothelial superoxide production. These studies demonstrated increased levels of oxygen radicals that were inhibited by ~20% by L-NAME. L-NAME is currently the only known specific inhibitor of NOS-derived superoxide production; however, this observation is based primarily on studies from purified enzyme. Because L-NAME is a methyl ester and is subject to modification by cellular esterases, its intracellular kinetics on NOS-derived superoxide production are not well characterized. Nevertheless, increased endothelial superoxide production was observed form BAECs exposed to 4-HNE; however, the source of this radical generation is unclear.

To determine whether the increased levels of ADMA observed following 4-HNE exposure resulted from changes in the activity of the ADMA metabolizing enzyme DDAH, its activity was measured. Studies of DDAH activity demonstrated a 40% decrease in hydrolytic activity, suggesting that the mechanism for the observed 4-HNE-directed NOS impairment was via an inhibition of DDAH. Additional studies were performed on purified recombinant hDDAH-1 to determine whether 4-HNE effects were through direct interaction with the enzyme. Results demonstrated that incubation of hDDAH-1 with 4-HNE (50 µM) resulted in a >40% decrease in enzyme activity. These effects were specific to 4-HNE as incubation with the nonoxidized carbonyl hexanol (10–500 µM) had no effect on DDAH activity. Similar studies were performed with purified recombinant eNOS and no inhibition was observed following 4-HNE exposure. 4-HNE forms Michael adducts with histindine and cysteine residues on proteins. In this regard, the catalytic triad of DDAH contains both cysteine and histidine residues, and mutation of either amino acid has been demonstrated to render the enzyme inactive (14, 37, 39).

As further support to the role of DDAH in mediating the inhibitory effects of 4-HNE on endothelial NO production, studies were performed using DDAH overexpressing BAECs. Overexpression of DDAH should lead to a decrease in cellular methylarginines with the concomitant increase in NOS-derived NO. DDAH overexpression was induced using an adenoviral construct carrying the human DDAH-1 gene (adDDAH1). Preliminary studies demonstrated that incubation of BAECs with adDDAH1 at 25 MOI, resulted in a threefold increase in protein expression and a >50% increase in DDAH activity following a 48-h incubation period. DDAH overexpression increased cellular DDAH activity in control cells by 50% and resulted in a 22% increase in cellular NO production (Figs. 6 and 7), demonstrating that the endogenous levels of ADMA and L-NMMA are sufficient to significantly inhibit endothelial NO generation. If one then considers the twofold increase in the levels of ADMA observed following the 4-HNE treatment, a ~40% inhibitory effect would be predicted (5). These data support previous studies implicating ADMA in the "arginine paradox" (1). The arginine paradox is a phenomenon in which supplementation with exogenous L-Arg enhances NO-mediated vascular function despite the fact that endothelial L-Arg concentrations are 50- to 100-fold above the Km. These results, for the first time, directly demonstrate that the endogenous methylarginines are able to basally regulate endothelial NO and implicate the methylarginines as direct mediators of the "arginine paradox," acting as competitive inhibitors of NOS and increasing the effective Km for L-Arg.

Subsequently, a series of studies were performed using this same transduction protocol to examine the effects of DDAH overexpression on 4-HNE-mediated endothelial NO inhibition. Although DDAH overexpression did increase DDAH activity and decrease endogenous methylarginines, the overexpression of the enzyme alone was not sufficient to prevent the 4-HNE-induced decrease in NO production. In fact, our results demonstrated that exposure of DDAH-overexpressing cells to 4-HNE resulted in worsened outcome as NO levels were significantly lower than that in the control cells exposed to 4-HNE. Although these results may appear contradictory to our hypothesis, they in fact support it and demonstrate that NOS uncoupling is likely occurring. We have previously demonstrated that ADMA inhibits NOS-derived superoxide, and as such, DDAH overexpression in the presence of uncoupled NOS would be expected to eliminate ADMA and thus prevent ADMA-mediated inhibition of NOS-derived superoxide. The outcome of this would be reduced NO bioavailability through the reaction of available NO with superoxide, a reaction that occurs at diffusion limited rates.

Our hypothesis would predict that treatment of DDAH-overexpressing cells with an antioxidant would restore NO to levels similar to those observed with L-Arg and GSH treatment, if in fact, methylarginines are contributing to the inhibition in NO generation seen with 4-HNE challenge. Indeed, we have demonstrated almost complete protection of cellular NO production following 4-HNE challenge using a combination of viral overexpression of DDAH and treatment with GSH, compared with the respective control. These results would indicate that GSH alone reduces NOS uncoupling but not the methylarginine accumulation, whereas L-Arg supplementation and/or DDAH overexpression overcomes the 4-HNE-induced increase in methylarginines but not the NOS uncoupling.

In conclusion, our results demonstrate for the first time that the lipid peroxidation product 4-HNE can inhibit the endothelial NO production. The doses used in this study represent pathological levels of this highly reactive lipid peroxidation product and suggest that this bioactive molecule may play a critical role in the endothelial dysfunction observed in a variety of cardiovascular diseases. The inhibitory effects of 4-HNE appear to be mediated through both oxidant stress and elevated levels of the endogenous NOS inhibitors ADMA and L-NMMA, as either L-Arg supplementation or DDAH overexpression in the presence of an antioxidant were able to restore NO production. Furthermore, DDAH-overexpressing cells exhibited significantly increased levels of NO and implicate the methylarginines as mediators of the "L-arginine paradox." Together, these results represent a major step forward in our understanding of the regulation, impact, and role of methylarginines and lipid peroxidation in cardiovascular disease.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant HL-081734 (to A. J. Cardounel).


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. J. Cardounel, Davis Heart and Lung Research Institute, 473 W. 12th Ave., Columbus, OH 43210 (e-mail: cardounel-1{at}medctr.osu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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