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Circulation Research. 1995;77:510-518

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(Circulation Research. 1995;77:510-518.)
© 1995 American Heart Association, Inc.

Articles

Native Low-Density Lipoprotein Increases Endothelial Cell Nitric Oxide Synthase Generation of Superoxide Anion

Presented in part at the 66th Scientific Sessions of the American Heart Association, Atlanta, Ga, November 8-11, 1993, and in full at the International Symposium on Endothelium-Derived Factors and Vascular Protection, San Francisco, Calif, January 21-25, 1995.

Kirkwood A. Pritchard, Jr, Laura Groszek, David M. Smalley, William C. Sessa, Mingdan Wu, Patricio Villalon, Michael S. Wolin, Michael B. Stemerman

From the Department of Pathology (K.A.P, D.M.S.), CardioVascular Research Center, Medical College of Wisconsin, Milwaukee; the Departments of Experimental Pathology (L.G., M.W., P.V., M.B.S.) and Physiology (M.S.W.), New York Medical College, Valhalla; and Boyer Center for Molecular Medicine (W.C.S.), Yale University School of Medicine, New Haven, Conn.

Correspondence to Kirkwood A. Pritchard, Jr, PhD, Associate Professor of Pathology and Pharmacology and Toxicology, Medical College of Wisconsin, CardioVascular Research Center, Rm 494B, 8701 W Watertown Plank Rd, Milwaukee, WI 53226.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract To examine mechanisms by which native low-density lipoprotein (n-LDL) perturbs endothelial cell (EC) release of superoxide anion (O2-) and nitric oxide (NO), ECs were incubated with n-LDL at 240 mg cholesterol per deciliter for 4 days with media changes every 24 hours. n-LDL increases EC release of O2- by more than fourfold and increases nitrite production by 57%. In the conditioned media from day-4 incubations, n-LDL increases total nitrogen oxides 20 times control EC (C-EC) levels. However, n-LDL did not alter EC NO synthase (eNOS) enzyme activity as measured by the [3H]citrulline assay. N{omega}-Nitro-L-arginine methyl ester, a specific inhibitor of eNOS activity, increases C-EC release of O2- by >300% but decreases LDL-treated EC (LDL-EC) release by >95%. L-Arginine inhibits the release of O2- from LDL-ECs by >95% but did not effect C-EC release of O2-. Indomethacin and SKF 525A partially attenuate LDL-induced increases in O2- production by {approx}50% and 30%, respectively. Thus, n-LDL increases O2- and NO production, which increases the likelihood of the formation of peroxynitrite (ONOO-), a potent oxidant. n-LDL increases the levels of nitrotyrosine, a stable oxidation product of ONOO-, and tyrosine by {approx}50%. In spite of this increase in oxidative metabolism, analysis of thiobarbituric acid substances reveals that no significant changes in the oxidation of n-LDL occur during the 24-hour incubations with ECs. These data indicate that n-LDL directly perturbs endothelial oxidative metabolism and uncouples L-arginine metabolism from NO release to increase eNOS generation of O2-. Such changes may represent one of the earliest EC perturbations in atherogenesis.


Key Words: low-density lipoprotein • reactive oxygen species • atherogenesis • peroxynitrite • superoxide anion peroxynitrite


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The relation between elevations in n-LDL concentrations and the premature development of atherosclerosis is well established. One of the primary defects in this disease process involves EC dysfunction characterized by diminished EDRF activity. Several groups have put forth the hypothesis that plasma cholesterol levels increase superoxide anion (O2-) production by the vessel wall, which inactivates EDRF.1 2 3 4 Indeed, threefold increases in this reactive species coincide with decreases in vascular relaxation during hypercholesterolemia, suggesting that inhibiting EDRF activity may play an important role in atherosclerosis.3 Although such changes may be fundamentally related to the dysfunctional states exhibited by the endothelium after exposure to n-LDL, little is known about the direct role of n-LDL in altering eNOS function.

n-LDL enhances EC prostacyclin production,5 P450-dependent generation of epoxyeicosatrienoic acids,6 endocytic activity,7 and permeability.8 In addition, n-LDL increases EC recruitment of mononuclear cells by perturbing cellular membrane lipid dynamics9 and the synthesis of adhesion molecules.10 These in vitro observations closely resemble many of the early changes observed in vivo and ex vivo in humans and animals during hypercholesterolemia.11 12 13 14

To determine the effects of n-LDL on EC reactive oxygen species generation, human umbilical vein ECs were exposed to n-LDL at 240 mg cholesterol per deciliter for 4 days, and changes in O2- and NO production were examined. n-LDL–induced changes in O2- release were assessed by the ferricytochrome c assay.15 The effects of n-LDL on NO release were examined by measuring changes in nitrite and nitrate levels by use of the Griess assay (Sessa et al16 ). Our findings indicate that n-LDL stimulates NO production and perturbs the endothelium to increase eNOS-dependent O2- production.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
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 down arrowDiscussion
 down arrowReferences
 
Materials
The reagents used for the studies were obtained as follows: medium 199 with Earle's salts, HEPES free acid, FBS, antibiotics/mycotics, heparin, potassium bromide (KBr), ferricytochrome c, SOD, sodium nitrite (NaNO2), L-arginine, L-NAME, phosphotungstate-MnCl2 precipitating reagent (catalogue No. 352-4), sucrose, Nonidet P-40, DTT, leupeptin, aprotinin, soybean trypsin inhibitor, PMSF, NADPH, CaCl2, and glucose from Sigma Chemical Co; sulfanilamide and NEDA from Aldrich; thymidine, fungizone, and DPBS) from GIBCO); Primeria 100-mm dishes and T75cm2 and T25cm2 flasks from Becton Dickinson-Falcon; six-well cluster plates from Corning; 24x30-mm Thermanox coverslips, eight-well chamber plates, and two chamber slides from Lux; human fibronectin from NY Blood Center; nonfrozen plasma from Hudson Valley Blood Services and Blood Center of Southeastern Wisconsin; EDTA from Fisher; BHT from Kodak; BH4 from Cayman Chemical Co; and, [3H]L-arginine (36.8 Ci/mol) from Dupont NEN.

Methods
EC Culture
Human umbilical vein ECs were obtained and passaged as described previously.6 9 17 ECs were maintained in medium 199 containing 16.7% FBS, 20 mmol/L HEPES, pH 7.4, antibiotics/mycotics, and 10 ng/mL r-bFGF). r-bFGF was kindly provided by John Anthony Thompson (University of Alabama, Birmingham). For all studies, ECs were used at passages 3 to 5 and grown to confluence before n-LDL exposure. ECs were passaged onto fibronectin-coated 24x30-mm Thermanox coverslips in eight-well chambers, six-well cluster plates, and two chamber slides for O2-, nitrite, and nitrotyrosine assays, respectively. Cell counts were performed on a Coulter counter (model Zf).

Isolation of LDL and Characterization
Nonfrozen human plasma from two to five donors was obtained, and BHT, a lipid-soluble antioxidant, and EDTA were immediately added to the plasma to achieve final concentrations of 20 µmol/L and 0.01%, respectively. The plasma was then mixed for 15 minutes at 4°C before ultracentrifugation. Sterile techniques, reagents, and dialysis solutions were used for isolation of n-LDL by sequential density ultracentrifugation (1.019 to 1.063 g/mL) as described previously.6 9 17 Cholesterol levels were determined by using a cholesterol oxidase colorimetric kit from Sigma. Endotoxin levels were determined by the colorimetric limulus amebocyte kit from BioWhitaker, as described previously.17 n-LDL was stored at 4°C and used for experiments within 2 weeks. This EC model of hypercholesterolemia was specifically designed to examine the effects atherogenic concentrations of n-LDL on EC function.5 6 7 9 10 17 ECs have been incubated with n-LDL in concentrations that exceed 160 mg cholesterol per deciliter for 4 days, with media changes every 24 hours.6 9 10 17 Previous studies demonstrated that the protocols for protecting n-LDL with BHT essentially eliminated nonspecific oxidation of the LDL particle during isolation and culture.6 17 In the present study, the oxidation state of an n-LDL particle, conditioned by standard culture with and without ECs, was characterized by TBARS assay. TBARS analysis was performed on conditioned C-EC and LDL-EC media as described previously5 6 17 by first precipitating apolipoprotein B containing lipoproteins from the media with phosphotungstate-MnCl2. MDA equivalents were quantified on a Cytofluor II (PerSeptive Biosystems). TBARS data were expressed as nanomoles MDA equivalents per milligram cholesterol.

Superoxide Anion Measurements
n-LDL–induced changes in O2- release were determined by following increases in ferricytochrome c absorbance at 550 nm on a Guilford spectrophotometer.15 ECs on coverslips were removed from the eight-well chamber with sterile forceps, cut to 20x30 mm, and washed three times in three changes of DPBS (50 mL). Two washed and trimmed coverslips were placed into a 20x30-mm quartz cuvette with the cells facing inward. DPBS (1.8 mL) was gently pipetted into the cuvette to minimize disturbing the EC monolayers. Ferricytochrome c (final concentration, 50 µmol/L) was added directly to the cuvette, the solution was mixed by gentle inversion, and changes in absorbance were followed for 15 minutes. Rates of O2- production were calculated on the basis of the molar extinction coefficient of reduced ferricytochrome c [{varepsilon}=21 000 cm-1 · (mol/L)-1] and the portion that was inhibited by SOD (400 U/mL). Cells counts were determined for calculating results as micromoles O2- per 106 cells per minute.

To rule out the possibility that changes in O2- release develop from nonspecific reduction of ferricytochrome c, several conditions were examined. Changes in ferricytochrome c reduction were observed in the DPBS alone and also when n-LDL and ox-LDL were added directly to the ferricytochrome c/DPBS buffer. As for the EC studies above, ferricytochrome c was added directly to DPBS at a final concentration of 50 µmol/L, mixed by inversion, and placed into the spectrophotometer, and changes in absorbance were read at 550 nm for 15 minutes.

To determine the effects of L-arginine on EC O2- release, an aliquot of L-arginine (in DPBS) was pipetted directly into the assay solution (final concentration, 250 µmol/L) and mixed by inversion immediately before monitoring the changes in absorbance.

To determine which oxidative enzyme systems contribute to the release of O2- after n-LDL exposure, C-ECs and LDL-ECs were incubated in their respective media with L-NAME (final concentration, 100 µmol/L), indomethacin (final concentration, 10 µmol/L), or SKF 525A (final concentration, 30 µmol/L) for 15 minutes. The coverslips were removed and prepared for analysis as described above. All assays were carried out with the inhibitor present in the assay solutions at same concentration used for preincubation.

Total Nitrogen Oxides and Nitrite Measurements
Changes in EC production of NO were assessed by determining nitrite levels by the Griess reaction.16 18 19 Briefly, C-ECs and LDL-ECs were washed three times with 3 mL DPBS and then incubated with 2 mL DPBS in a 37°C oven purged with a gas mixture of 5% CO2/95% air and rotated at 80 rpm for 30 minutes. To determine the effectiveness of L-NAME inhibition of eNOS activity, C-ECs and LDL-ECs were incubated with L-NAME (100 µmol/L) in their respective media for 15 minutes. The cells were then washed and incubated with DPBS containing the 100 µmol/L L-NAME as described above for the O2- assay. Analysis was performed on a single aliquot from each well. Briefly, a 1 mL mixture of sulfanilamide (final reaction concentration, 0.3% [wt/vol]) and NEDA (final reaction concentration, 0.03% [wt/vol]) was added to 0.8 mL of the conditioned DPBS, vortexed, and then incubated for 10 minutes at 37°C. The absorbance of the resulting pink chromophore from the unknowns was read at 550 nm and compared with the absorbance values obtained from standard NaNO2 solutions (0 to 200 µmol/L).

To assay for total NOx, nitrate was first converted to nitrite with an aliquot of Escherichia coli (American Type Culture Collection 25922), which was grown anaerobically to enrich nitrate reductase activity as described by Ischiropoulos et al.20 For these studies, reagents to sample volumes were adjusted so that the assay could be performed in microtiter plates. The final reagent concentration to sample volume, however, remained the same. The Griess reaction for nitrite was then carried out on aliquots of EC-conditioned media, which were preincubated with nitrate reductase–enriched E coli for 70 minutes.

eNOS Activity Measurements: The Arginine-Citrulline Assay
The effects of n-LDL on eNOS metabolism of [3H]arginine to [3H]citrulline were determined as described previously.21 22 23 EC lysates were made from C-EC and LDL-EC cultures as described previously.21 22 23 The assay was as follows: ECs were suspended in cold lysis buffer (0.3 mol/L sucrose, 10 mmol/L HEPES, 1% Nonidet P-40, 0.1 mmol/L EDTA, 1 mmol/L DTT, 10 µg/mL leupeptin, 2 µg/mL aprotinin, 10 µg/mL soybean trypsin inhibitor, and 50 µmol/L PMSF, pH 7.4), vortexed, and then kept at -80°C until analysis. Cell lysates (150 to 250 µg protein) were combined with NADPH (2 mmol/L), CaCl2 (230 µmol/L), BH4 (3 µmol/L), and [3H]L-arginine (0.2 µCi, 10 µmol/L) for 20 minutes at 37°C. The assay volume was kept constant at 100 µL. To determine if n-LDL altered iNOS activity, the assay was repeated with EDTA (1.7 mmol/L) replacing calcium in the assay buffers. iNOS is calcium insensitive, whereas eNOS is calcium sensitive for activation.

Nitrotyrosine Measurements by Enzyme-Linked Immunoassay
ECs were grown to confluence in two chamber slides and then incubated with n-LDL for 4 days (240 mg cholesterol per deciliter). C-ECs and LDL-ECs were washed three times with DPBS and then fixed in 3.7% formaldehyde in DPBS for 20 minutes. Cell membranes were made permeable with 0.5% Triton X-100 in PBS for 15 minutes to allow the antibodies access to intracellular proteins. Monoclonal nitrotyrosine antibodies (kindly provided by Joseph Beckman, University of Alabama, Birmingham) were diluted 1:50 in 1% BSA/DPBS and incubated with the cells at room temperature for 1 hour. The slides were then washed three times with BSA/DPBS. Next, a 1:250 dilution of anti-mouse IgG antibody linked to horseradish peroxidase (Biorad) was added and incubated at room temperature for 30 minutes. The slides were washed three times with DPBS and then incubated with OPD/H2O2 in an acetate buffer (pH 5) for 30 minutes. The reaction was stopped by adding 50 µL of 2N H2SO4. A 200-µL aliquot of the OPD reaction mixture was transferred to an ELISA plate, and absorbances were read at 492 nm. As a positive control for peroxynitrite(ONOO-)–induced nitrotyrosine formation, a second set of washed control ECs were incubated with authentic ONOO- (2x25 µmol/L).24 Each test group, on a separate slide, was incubated with excess nitrotyrosine (10 µmol/L) to competitively block binding of the nitrotyrosine antibody to cell-associated nitrotyrosine. Nonspecific antibody binding was assessed by adding a nonspecific mouse monoclonal IgG antibody (MOPC-31C, Sigma Chemical Co) in place of the anti-nitrotyrosine antibody. Results are expressed as the percentage of C-EC nitrotyrosine levels.

Statistical Analysis
Data represent the mean±SEM in the table and all figures, unless otherwise indicated. Statistical analysis was by the one-tailed Student's t test. The level of significance was set at P<.05. Questions concerning the significance of treatments were constructed to determine whether treatments resulted in significant reductions or enhancements of analyte production.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
To examine the effects of atherogenic concentrations of n-LDL on EC function, it was necessary to develop isolation and culture protocols that essentially eliminated the possibility of oxidative modification of the LDL particle.5 6 17 Adding BHT to the plasma and to the sterile saline dialysis solutions is sufficient for protecting n-LDL from the oxidation observed when LDL is not protected with antioxidants.25 26 The TableDown illustrates the changes in TBARS levels in n-LDL in the media incubated in the absence and presence of ECs. TBARS analysis demonstrates that the BHT-protected n-LDL particles experience little change in oxidation when incubated alone or with ECs. ox-LDL is used as a control for comparison purposes and contains 6.92 nmol MDA per milligram cholesterol. Furthermore, n-LDL prepared under these protocols contains levels of endotoxin that are well below those required for activation (<0.01 EU/mL).17 Taken together, these data demonstrate that n-LDL can be used in tissue culture for examining the effects of hypercholesterolemia on EC function. The protocols described here provide n-LDL in sufficient quantities and quality to allow one to examine perturbations in EC biochemistry before the effects of oxidized products of altered LDL.


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  		Table 1. BHT Protects n-LDL Against Oxidation

Using this model of hypercholesterolemia, we found that the n-LDL increase in EC release of O2- is more than four times the levels released by C-ECs (Fig 1Down). Results in Fig 1Down are corrected to reflect only that portion of O2- production that is blocked by SOD (400 U/mL). The majority of O2- signal from both C-ECs and LDL-ECs is blocked by SOD (C-EC+SOD, 0.026±0.004 µmol O2- per minute per 106 cells; LDL-EC+SOD, 0.031±0.006 µmol O2- per minute per 106 cells). Furthermore, BHT did not appear to interfere with the ability of endothelium to release O2-. Thus, atherogenic concentrations of n-LDL induce profound changes in the oxidative metabolism of the endothelium. No increases in ferricytochrome c absorbances were noted in DPBS or when n-LDL (20 µg protein per milliliter) or ox-LDL (20 µg protein per milliliter) was added to the assay buffers (data not shown). Our findings demonstrate that the increases in O2- release are the direct result of n-LDL effects on the endothelium and are not due to an artifact of ox-LDL in the assay system.



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  		Figure 1. Native LDL increases eNOS-dependent O2- generation. ECs increase SOD-inhibitable O2- generation after 4 days of n-LDL exposure (240 mg cholesterol per deciliter). Enzymatic sources for O2- release were probed with L-arginine (L-ARG, 250 µmol/L), L-NAME (100 µmol/L), indomethacin (INDO, 10 µmol/L), and SKF 525A (SKF, 30 µmol/L). L-ARG significantly reduces LDL-induced O2- release (P<.01) while having no effect on C-EC release. L-NAME significantly increases C-EC O2- release (P<.05) but decreases LDL-EC culture O2- release (P<.01), indicating that in LDL-EC cultures eNOS may be an important source of O2-. Inhibition of cyclooxygenase activity with indomethacin and P450 isozyme activity with SKF resulted in partial but statistically significant reductions in LDL-EC O2- generation (P<.05).

Several mechanisms are proposed to explain increases in O2- production of the vessel wall during hypercholesterolemia.3 4 27 Loss of functional NO is examined by including L-arginine or L-NAME in the buffers of the ferricytochrome c assay. L-Arginine (250 µmol/L) does not significantly alter C-EC release of O2- (Fig 1Up). In contrast, L-arginine markedly decreases the release of O2- from LDL-ECs to the levels obtained with SOD or L-NAME. Since L-arginine does not inhibit xanthine/xanthine oxidase reduction of ferricytochrome c,28 it is likely that reductions in LDL-EC O2- release develop from uptake and metabolism of this amino acid to NO, which in turn scavenges O2-. Alternatively, excess L-arginine may keep the heme site of eNOS fully occupied to limit uncoupled O2- generation. Regardless of the mechanism, adding L-arginine reduces O2- production rates by LDL-ECs below the levels observed for C-ECs.

To determine whether NO plays equivalent roles in C-EC and LDL-EC cultures, the effects of L-NAME, a specific inhibitor of NOS activity, on O2- release are assessed. L-NAME increases C-EC release of O2- by >300% (Fig 1Up). These data are consistent with the notion that NO functions as an intracellular scavenger of O2- and that decreases in functional NO increase the release of O2-. In contrast, L-NAME reduces LDL-EC release of O2- by >95% (Fig 1Up). According to our previous studies, n-LDL increases COX and P450 activities,6 29 which are well-recognized sources of O2- production.30 To examine the contribution of these pathways, C-ECs and LDL-ECs are incubated with indomethacin (10 µmol/L) and SKF 525A (30 µmol/L). Indomethacin and SKF 525A reduce LDL-EC release rates by {approx}50% and 30%, respectively (Fig 1Up). However, it is important to note that L-NAME is more effective in inhibiting LDL-EC O2- release than are indomethacin and SKF 525A (Fig 1Up). Thus, protracted EC exposure to atherogenic n-LDL concentrations increases O2- production by three independent oxidative enzyme systems. More important, eNOS appears to be the greatest source among these pathways for n-LDL-induced increases in O2- production.

To determine whether n-LDL alters the usual generation of NO, the levels of NOx released into 24-hour conditioned culture media from day 4 and the levels of nitrite released into 30-minute conditioned DPBS were determined.16 18 19 During culture in medium 199, which contains 330 µmol/L L-arginine, n-LDL increases NOx levels by 20-fold (Fig 2ADown). In addition, in the absence of L-arginine, NO2- production rates by washed LDL-ECs are significantly increased (Fig 2BDown), although the relative increase is much smaller than when ECs are exposed to media containing both n-LDL and L-arginine (Fig 2ADown). BHT did not appear to inhibit the ability of endothelium to generate NOx or NO2-. In our hands, adding L-NAME to cultures inhibits formation of {approx}50% of the chromophore detected by the Griess reaction (data not shown).



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  		Figure 2. LDL activates eNOS. LDL-EC cultures release significantly more total NOx into the conditioned media during a 24-hour exposure than do C-ECs. A, Increase in levels of NOx in the conditioned media from day-4 samples. B, Levels of nitrite released by washed cells incubated in DPBS. Washed LDL-ECs release significantly higher levels of nitrite in 30 minutes than do C-ECs (for media, n=12, C-ECs vs LDL-ECs, P<.01; for DPBS, n=6, C-ECs vs LDL-ECs, P<.025). Please note that data in panel A are expressed as micromolar values and data in panel B are expressed as rates (nanomoles per 106 cells per minute).

The effects of n-LDL on eNOS activity are examined by the [3H]citrulline assay, which is performed under apparent Vmax conditions.21 22 23 n-LDL did not significantly alter the ability of eNOS in lysed cell preparations to metabolize L-arginine to L-citrulline (81.4±11.7 [C-ECs] versus 56.8±15.3 [LDL-ECs] pmol citrulline per milligram protein per minute, n=4, mean±SD). Furthermore, the activity of iNOS, a calcium-independent enzyme, in these cell preparations is sufficiently low ({approx}0.45 pmol citrulline per milligram protein per minute) that it is unlikely that it could account for n-LDL–induced increases in reactive oxygen species generation.

Dual increases in O2- and NO are the prerequisite conditions for increasing ONOO- formation.31 32 ONOO- oxidizes tyrosine to nitrotyrosine, which can be detected immunogenically.4 32 To determine the effects of n-LDL on ONOO- formation, nitrotyrosine levels were measured by using antibodies kindly provided by Joseph Beckman (Birmingham, Ala). n-LDL significantly increases immunoreactive nitrotyrosine levels by {approx}50% above C-EC levels. The nitrotyrosine levels in LDL-EC cultures after 4 days are {approx}33% of the levels formed after exposing C-ECs to two doses of authentic ONOO- (Fig 3Down).



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  		Figure 3. n-LDL increases peroxynitrite (ONOO-) formation: changes in nitrotyrosine (NT) content. C-ECs and LDL-ECs were examined for changes in immunoreactive nitrotyrosine levels by the ELISA-OPD assay described in "Methods." Protracted exposure to n-LDL significantly increases EC nitrotyrosine content by {approx}50% relative to C-ECs (100%) (P<.05). NT (10 µmol/L) was added to C-ECs and LDL-ECs to block specific binding of the antibody to EC cultures. Authentic ONOO- was added to C-ECs as positive control for ONOO--mediated nitrotyrosine formation. ONOO- increased C-EC nitrotyrosine levels nearly threefold. Results are expressed as percentage of C-EC nitrotyrosine levels (n=3, C-ECs vs LDL-ECs, P<.01).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The present study demonstrates that pathophysiological concentrations of n-LDL perturb EC oxidative metabolism to increase O2- production. NO plays an important role in limiting the amount of O2- released by ECs, as illustrated by L-NAME–induced increases in O2- release from C-ECs. However, inhibition of O2- release from LDL-ECs by L-NAME points to a mechanism by which n-LDL perturbs EC function to increase eNOS generation of O2-. Selective inhibition of COX and P450 isozymes attenuates increases in O2- release induced by n-LDL but not to the extent that L-NAME inhibits O2-. Thus, n-LDL increases oxidative metabolism by several enzyme systems. However, of the enzyme systems examined, eNOS appears to generate the largest portion of the O2-. This conclusion is supported by decreases in O2- release from LDL-ECs but not C-ECs with 250 µmol/L L-arginine. These data suggest that in LDL-EC cultures L-arginine metabolism may be impaired. Further, uncoupling L-arginine metabolism from NO release is probably a critical step in eNOS-dependent O2- generation. The ability of the endothelium to produce NO is not compromised, since n-LDL, in the presence of L-arginine, stimulates increases in NO production. Furthermore, the conversion of [3H]arginine into [3H]citrulline under apparent Vmax conditions, a measure of eNOS levels, is not significantly different between LDL-EC and C-EC lysates. BHT did not appear to inhibit O2- and NOx synthesis, since rates of production of these two analytes were not impaired. Dual increases in the generation of O2- and NO are the principal and prerequisite conditions for ONOO- formation, which is increased in LDL-ECs. Thus, protracted n-LDL exposure induces EC dysfunction such that eNOS becomes a new source for O2- generation and ONOO- formation. These studies link atherogenic concentrations of n-LDL with increases in O2- generation from altered eNOS activity.

The central hypothesis of our laboratories is that n-LDL directly alters endothelial function.5 6 7 9 17 Previous studies indicate that n-LDL prepared by our methods does not experience significant increases in oxidation.6 17 Thus, adding BHT to the plasma before n-LDL isolation is sufficient to limit oxidation during culture. Protecting n-LDL against oxidation is the first step in being able to examine perturbations in EC function induced by n-LDL rather than by ox-LDL. Our new findings extend previous studies by demonstrating how n-LDL uncouples L-arginine metabolism from eNOS activity, which in turn alters one of the critical functions of the endothelium generation of EC-derived NO. n-LDL uncouples L-arginine metabolism from eNOS generation of NO, resulting in increased O2- and ONOO- production. Under these conditions, functional NO levels are probably diminished as well. Thus, n-LDL–exposed ECs will experience not only increases in O2- production but also decreases in NO. Such perturbations in usual reactive oxygen species generation may play an important role in the activation of oxidant-sensitive transcription factors, ie, nuclear factor-{kappa}B and fos/jun, which in turn influence the expression of adhesion molecules.33 34 35 Interestingly, when L-NAME is used to inhibit NO production in human endothelial cells, intercellular adhesion molecule-1–dependent binding of neutrophils is increased.36 The effects of n-LDL on O2- and ONOO- generation may explain, in part, the mechanisms by which n-LDL increases endothelial adherence of mononuclear cells observed by us in vitro9 17 and by Tsao et al28 ex vivo. Interestingly, L-arginine suppression of O2- release from LDL-EC cultures reveals new insight into a possible mechanism by which L-arginine supplementation of cholesterol-fed rabbits decreases mononuclear adherence.28

The relative levels of NO generation by cells of the vessel wall are critical for proper function of the vasculature. During hypercholesterolemia, the vessel wall experiences changes in the balance of oxidative and antioxidative enzyme systems, resulting in increases in O2- release.3 37 Early in the disease state, this increase is manifested in the endothelium3 ; later, it is predominantly associated with the monocytes/macrophages, which release reactive oxygen species in much larger concentrations than ECs.20 38 Inhibition of eNOS with arginine analogues not only compromises endothelium-dependent mechanisms of relaxation but also perturbs the balance between oxidation and reduction.28 39 40 41 Such changes appear intimately linked to increases in plaque development.28 39 40 41 From the above studies, one can conclude that NO functions as an antiatherogenic molecule, as suggested by Cooke and Tsao.42

Recent evidence indicates that when cellular levels of O2- and NO are increased, the potent oxidant, ONOO-, is quickly formed.20 31 32 ONOO- is described as a "cloaked oxidant," which exhibits a wide range of oxidative cytotoxic properties.31 Its pathophysiological importance was recently demonstrated by using nitrotyrosine antibodies. In these studies, advanced plaques were found to contain high levels of nitrotyrosine, implicating involvement of ONOO- in late- to end-stage lesion formation.4 43

ONOO- formation and its relevance to atherosclerosis should not, however, be limited to mononuclear type cells. When ECs are stimulated with ionophore, marked increases in ONOO- are observed.44 45 Our data indicate that protracted EC exposure to atherogenic LDL concentrations significantly increases EC production of ONOO- during basal metabolic activity. Thus, ONOO--mediated atherogenic mechanisms are probably invoked before the oxidant injury mechanisms in advanced lesions. These findings link n-LDL with increases in endothelial ONOO- generation. Accordingly, such changes will play critical roles in endothelial activation mechanisms that are oxidant stress sensitive.

Increases in NO production induced by n-LDL indicate that this lipoprotein acts as a stimulant for eNOS activity. Increases in ONOO- induced by n-LDL result from NO condensing with O2-, an event that not only lowers functional NO levels but also increases the formation of a new oxidant with properties different from either O2- or NO. These in vitro findings support the concept that although hypercholesterolemia increases NO production early in the atherogenic process, the NO that is produced is ineffective.27 Because [3H]citrulline assays are performed under apparent Vmax conditions, the conversion rates are representative of enzyme levels. Although rates of conversion in LDL-EC lysates are slightly less than the rates in C-EC lysates, these differences are not significant. Together, these data suggest that n-LDL may perturb either substrate delivery or the availability of a cofactor to promote eNOS generation of O2-. It should be noted that the ferricytochrome c assay did not include L-arginine, a condition that is not optimal for eNOS activity. Perhaps the absence of L-arginine magnifies defects in eNOS function, leading to increases in O2- release from LDL-ECs. In vivo evidence that eNOS can be uncoupled to increase oxidant stress is found in rabbits fed diaminohydroxypyrimidine to block synthesis of BH4.46 This treatment results in aortas that produce higher levels of H2O2, a product of O2- dismutation, than do control aortas. Finally, the effects of n-LDL on eNOS are opposite those of LDL that oxidizes during culture26 and ox-LDL47 but are consistent with increases in NOx production by aortas from cholesterol-fed rabbits.27 Thus, n-LDL uncoupling of eNOS could be viewed as a mechanism that occurs early in the atherogenic process, whereas the effects of ox-LDL could be viewed as a series of mechanisms that occur after LDL enters the vessel wall and becomes trapped and then modified.

L-NAME and L-arginine inhibition of eNOS O2- generation are both significant and complementary. One agent blocks the activity of eNOS; the other supplies excess substrate for generating NO, which in turn scavenges O2-.27 48 Indomethacin and SKF 525A do not lower LDL-EC O2- production to control levels, because eNOS continues to generate O2-. The levels of indomethacin and SKF 525A used here completely block arachidonic acid metabolism by COX and P450 isozymes, respectively,6 but do not alter eNOS activity. L-NAME does not inhibit the metabolism of arachidonic acid by COX and P450 isozymes (authors' unpublished observations, 1994). On the basis of the specificity of L-NAME and L-arginine for eNOS and the fact that reciprocal changes in O2- release are observed after treatment, it is unlikely the increase in O2- is derived from non-eNOS sources that are sensitive to both agents in the same way. We acknowledge that our data do not exclude such a possibility but consider it to be extremely remote.

At first glance, data in Fig 1Up may appear inconsistent, since inhibitions by L-NAME, indomethacin, and SKF 525A are greater than 100%. The reason this occurs is not clear. Extracellular release of O2- from ECs represents the sum totals of generation and scavenging as well as the effects of compartmentation and diffusion. Further, changes in NO and O2- production by eNOS after n-LDL treatments may confer unknown effects on COX and P450 activities. For example, NO increases COX prostaglandin production49 and inhibits P450 activity.50 Thus, NO from L-arginine may play a role in increasing O2- by enhancing COX activity. On the other hand, loss of functional NO, which likely occurs in LDL-ECs, may allow P450-dependent O2- production to increase, because NO is not available to inhibit the heme site of P450 isozymes. However, the effects of NO on O2- generation from these two sources are not well studied. Finally, LDL-induced increases in O2- and ONOO- may enhance the conversion of xanthine dehydrogenase to xanthine oxidase to increase O2-.51 This possibility may explain the mechanism by which xanthine oxidase in the aortas of cholesterol-fed rabbits became a source of O2- production.3 Unfortunately, because of the complexities of the interaction of NO with COX and P450 (and possibly other oxidative enzymes) it may not be possible to sort out the exact contribution each oxidative pathway makes by using inhibitors.

Another problem facing studies designed to probe the role of oxidative enzymes in reactive oxygen generation is inhibitor specificity. Indomethacin and SKF 525A are considered selective inhibitors of COX and P450 isozymes, yet these agents may alter EC function in ways that go beyond their direct effects on enzyme activity. For instance, it has been reported that indomethacin is capable of scavenging O2- and that SKF 525A is capable of perturbing calcium homeostasis.52 53 For our studies, we chose concentrations of indomethacin and SKF 525A that were previously shown not to scavenge O2-38 ; accordingly, the decreases in O2- detected should represent the relative contribution these pathways make to the total signal induced by n-LDL. Inhibitor targeting of other oxidative enzymes also has its problems. For example, diphenylene iodonium, a selective inhibitor of NADPH oxidoreductase, also inhibits NOS.54 Accordingly, the effects of this agent on both enzymes would further confuse the issue of which and how much each source contributes to n-LDL–induced increases in O2- generation. However, it should be noted that L-NAME is probably the most specific inhibitor of the agents used in the present study. Further, when L-NAME and L-arginine data are examined together, it strongly suggests that n-LDL uncouples eNOS activity to induce this enzyme to generate O2-.

The mechanisms by which L-NAME and L-arginine analogues specifically modulate brain NOS O2- generation are in dispute at this time. Pou et al55 show that L-NAME but not NG-monomethyl-L-arginine blocks neuronal brain NOS O2- production as did Heinzel et al56 for neuronal NOS H2O2 production. Frey et al57 report that L-thiocitrulline, a new inhibitor of NOS activity, blocks nearly 85% of the O2- signal from neuronal brain NOS under uncoupling conditions. Possibly, some insight into the mechanisms by which L-NAME inhibits O2- production can be obtained from the knowledge that L-NAME binds within the active site of NOS and decreases electron flux by inhibiting the reduction potential of heme iron.58 These studies suggest that L-NAME inhibits NOS O2- generation because it prevents electron transfer to the heme site. L-NMMA inhibits NOS conversion of arginine to citrulline by occupying the active site but does not appear to block electron transfer to the heme site. If this is the case, then it is likely that the iron heme group becomes electron rich and transfers the electrons to molecular oxygen to generate O2-. An alternative mechanism for O2- generation from NOS, however, may involve BH4; this finding is based on work demonstrating that neuronal brain NOS, made deficient in BH4, concurrently generates O2-, H2O2, and NO when L-arginine is present in low levels, a finding that can be fully reversed by adding BH4.56 Regardless of the mechanisms by which NOS generates O2-, data presented here clearly indicate that n-LDL uncouples eNOS activity to increase O2- production in L-arginine–deficient buffers.

The mechanisms by which n-LDL perturbs eNOS function to increase O2- generation remain unclear. In vitro and in vivo studies suggest that uncoupled O2- generation may be a unique property of eNOS that is manifested under conditions of L-arginine depletion and/or lack of cofactors such as BH4. When electron transfer is uncoupled in oxygenase enzymes, oxygen becomes the usual electron acceptor, resulting in O2- generation.59 When L-arginine is left out of brain NOS assays, increases in O2- production are observed.55 In vitro, the reduction of ferricytochrome c by neuronal brain NOS, in the presence of calcium/calmodulin, can be substantially reduced by SOD.57 60 When L-arginine levels fall below 100 µmol/L, NADPH oxidation is uncoupled from NOS synthesis of NO,56 which will increase O2- production. Under other conditions, glutamate uncouples L-arginine metabolism from neuronal NOS NO generation and increases in O2- production.61 BH4-deficient NOS concurrently generates O2-, H2O2, and NO; however, when BH4 is provided, maximal NO production is restored.56 This last mechanism may explain the increase in H2O2 production in aortas from rabbits fed diaminohydroxypyrimidine,46 an inhibitor of GTP cyclohydrolase I, the first enzyme in the synthesis of BH4.62 Taken together, these reports suggest that uncoupling L-arginine metabolism from NOS activity leads to increased O2- in vitro and in vivo. Previously, we demonstrated that n-LDL increases EC membrane rigidity through donating cholesterol.9 Changes in membrane lipid dynamics might impede the binding, internalization, and delivery of L-arginine to eNOS. Hypothetically, if n-LDL enhances eNOS activity and impedes the process governing L-arginine delivery to NOS, then intracellular L-arginine concentrations surrounding NOS may become depleted. According to the information above, such a state might uncouple NOS activity and increase the generation of O2-. Thus, one potential mechanism by which n-LDL may be inducing eNOS to generate O2- is by decreasing membrane fluidity to perturb usual L-arginine metabolism. Other potential mechanisms for the effects of n-LDL include perturbations in BH4 metabolism and calcium/calmodulin interactions. Finally, n-LDL may perturb the basic enzymatic properties of eNOS such that this trifunctional enzyme exhibits different requirements for L-arginine, BH4, flavin adenine dinucleotide, flavin mononucleotide, and /or calcium/calmodulin.

In summary, our findings demonstrate that n-LDL alters the usual reactive oxygen species generation. More important, L-NAME and L-arginine inhibition of O2- release from LDL-EC supports the notion that n-LDL uncouples L-arginine metabolism from NO release, thereby allowing eNOS to become a new source of O2-. These findings provide new insight into the mechanisms by which n-LDL both stimulates NO production and decreases functional NO levels through the formation of ONOO- (L-arginine supplementation limits n-LDL–induced mechanisms of endothelial dysfunction) and demonstrate the importance of NO limiting O2- release. These studies demonstrate that protracted EC exposure to atherogenic concentrations of n-LDL uncouples L-arginine metabolism from NO production and increases eNOS-dependent O2- generation and ONOO- formation. Finally, such perturbations may be central to n-LDL–induced mechanisms of EC dysfunction and atherogenesis.


*    Selected Abbreviations and Acronyms
 
BH4 = tetrahydrobiopterin
BHT = butylated hydroxytoluene
BSA = bovine serum albumin
C-EC = control EC
COX = cyclooxygenase
DPBS = Dulbecco's phosphate buffered saline
DTT = dithiothreitol
EC = endothelial cell
EDRF = endothelium-derived relaxing factor
ELISA = enzyme-linked immunosorbent assay
eNOS = EC NOS
FBS = fetal bovine serum
iNOS = inducible NOS
L-NAME = N{omega}-nitro-L-arginine methyl ester
LDL = low-density lipoprotein
LDL-EC = LDL-treated EC
MDA = malonyldialdehyde
n-LDL = native LDL
NEDA = N-(1-naphthyl)ethylenediamine dihydrochloride
NO = nitric oxide
NOS = NO synthase
NOX = nitrite+nitrate
OPD = orthophenylenediamine
ox-LDL = oxidized LDL
P450 = cytochrome P-450
PMSF = phenylmethylsulfonyl fluoride
r-bFGF = recombinant basic fibroblast growth factor
SOD = superoxide dismutase
TBARS = thiobarbituric acid reactive substances


*    Acknowledgments
 
This study was supported by National Heart, Lung, and Blood Institute grants HL-48521, HL-33742, HL-31069, and 1-PO HL-43023R1 and grant 93-006GB from the American Heart Association, New York State Affiliate, Inc. We gratefully acknowledge the gift of anti-nitrotyrosine antibodies kindly provided by Dr Joseph Beckman (University of Alabama, Birmingham) and extend our thanks to Drs Kalyanaraman, Hogg, and Griffith (Medical College of Wisconsin, Milwaukee) for their advice and comments in the writing of this manuscript. We also thank the staff of Labor and Delivery at White Plains Hospital (White Plains, NY) and Waukeshaw Memorial Hospital (Waukeshaw, Wis) for providing umbilical cords and Hudson Valley Blood Center (Valhalla, NY) and the Blood Center of Southeastern Wisconsin (Milwaukee) for providing plasma used for these studies. Finally, we thank Michelle Italiano for technical assistance in culturing of endothelial cells and n-LDL isolation.

Received September 26, 1994; accepted May 8, 1995.


*    References
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