The Binding of Oxidized Low Density Lipoprotein (ox-LDL) to ox-LDL Receptor-1 Reduces the Intracellular Concentration of Nitric Oxide in Endothelial Cells through an Increased Production of Superoxide*

Luciano CominaciniDagger, Anna Rigoni, Anna Fratta Pasini, Ulisse Garbin, Anna Davoli, Mario Campagnola, Antonio M. Pastorino, Vincenzo Lo Cascio, and Tatsuya Sawamura§

From the Department of Biomedical and Surgical Sciences, Verona University, 37134 Verona, Italy and § Department of Bioscience, National Cardiovascular Center Research Institute, Fujishirodai, Suita, Osaka, 565-8565, Japan

Received for publication, November 26, 2000, and in revised form, January 18, 2001




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Oxidized low density lipoprotein (ox-LDL) has been suggested to affect endothelium-dependent vascular tone through a decreased biological activity of endothelium-derived nitric oxide (NO). Oxidative inactivation of NO is regarded as an important cause of its decreased biological activity, and in this context superoxide (O&cjs1138;2) is known to inactivate NO in a chemical reaction during which peroxynitrite is formed. In this study we examined the effect of ox-LDL on the intracellular NO concentration in bovine aortic endothelial cells and whether this effect is influenced by ox-LDL binding to the endothelial receptor lectin-like ox-LDL receptor-1 (LOX-1) through the formation of reactive oxygen species and in particular of O&cjs1138;2. ox-LDL induced a significant dose-dependent decrease in intracellular NO concentration both in basal and stimulated conditions after less than 1 min of incubation with bovine aortic endothelial cells (p < 0.01). In the same experimental conditions ox-LDL also induced O&cjs1138;2 generation (p < 0.001). In the presence of radical scavengers and anti-LOX-1 monoclonal antibody, O&cjs1138;2 formation induced by ox-LDL was reduced (p < 0.001) with a contemporary rise in intracellular NO concentration (p < 0.001). ox-LDL did not significantly modify the ability of endothelial nitric oxide synthase to metabolize L-arginine to L-citrulline. The results of this study show that one of the pathophysiological consequences of ox-LDL binding to LOX-1 may be the inactivation of NO through an increased cellular production of O&cjs1138;2.




    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Endothelium-dependent relaxation is impaired in animals with atherosclerosis (1-3), which has been linked to a decreased production and/or biological activity of endothelium-derived nitric oxide (NO)1 (4, 5). Oxidative inactivation of NO is regarded as an important cause of its decreased biological activity (6). The vascular release of superoxide (O&cjs1138;2) radicals is sharply increased in atherosclerotic arteries (7, 8), and O&cjs1138;2 is known to inactivate NO in a chemical reaction during which the cytotoxic radical peroxynitrite is formed (9, 10). The presence of peroxynitrite-derived nitrotyrosines has recently been demonstrated in human atherosclerotic lesions (11).

Oxidized low density lipoprotein (ox-LDL) has been observed to induce abnormalities in endothelial function, which may be relevant for the progression of atherosclerotic lesions (12). In particular functional alterations of the endothelial cells may be involved in the reduction of vasodilation, in response to stimuli that induce NO release, in isolated arteries exposed to ox-LDL (13).

Recently, an endothelial receptor for ox-LDL, called lectin-like ox-LDL receptor-1 (LOX-1) was cloned from cultured bovine aortic endothelial cells (BAECs) (14). It has been suggested that ox-LDL uptake through this receptor may be involved in endothelial activation or dysfunction in atherogenesis (14). In this context we recently reported that ox-LDL binding to LOX-1 determined a significant increase in the generation of reactive oxygen species (ROS) in endothelial cells (15). In this report we investigated the relationship between the intracellular production of ROS and in particular of O&cjs1138;2 and the intracellular concentration of NO in cultures of BAECs exposed to ox-LDL.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

LDL Isolation-- Whole blood, obtained by venipuncture from healthy volunteers after 12 h of fasting, was collected into Vacutainer tubes (Becton Dickinson, Meylan, France) containing EDTA (1 mg/ml) and processed for LDL separation within 1 day by sequential flotation in NaBr solution (16) containing 1 mg/ml EDTA.

LDL Oxidation and Modification-- Cu2+-modified LDL (1.7 mg of protein/ml) was prepared by exposure of LDL to 5 µM CuS04 for 18 h at 37 °C as described previously (17, 18). The extent of LDL oxidation was determined by thiobarbituric acid-reactive substances as reported (18). Protein was measured by the Pierce BCA protein assay reagent (19). Malondialdehyde-modified LDL (MDA-LDL) was prepared according to a previously described method (20, 21). Acetylation of LDL was achieved by repeated additions of acetic anhydride (22).

Cell Cultures-- BAECs were isolated and cultured as described previously (23). Cells used for experiments were at passage levels between 2 and 4. Chinese hamster ovary-K1 (CHO-K1) cells and a CHO-K1 cell line stably expressing bovine LOX-1 (BLOX-1-CHO) (14) were cultured as described previously (23). Cell survival was monitored according to the method of Landegren (24).

ROS and O&cjs1138;2 Measurement-- Intracellular ROS production was monitored by following the oxidation of 2',7'-dichlorofluorescin diacetate in flow cytometry as described by Royall (25) and slightly modified by Zulueta (26). Intracellular O&cjs1138;2 generation was detected using a previously established flow cytometry technique based on the O&cjs1138;2-induced conversion of the oxidant-sensitive dye, hydroethidine (HE) to ethidium (27). BAECs were also tested for the production of H2O2 and superoxide radicals induced by ox-LDL. The amount of H2O2 released into the culture medium was determined fluorimetrically (28) and, that of O&cjs1138;2 was determined photometrically by reduction of cytochrome c (29).

Confluent BAECs in 24-well plates were incubated in Dulbecco's modified Eagle's medium (Sigma) containing 10% fetal bovine serum, 10 µM 2',7'-dichlorofluorescin diacetate (Eastman Kodak Co., Rochester, NY), or 1 µM HE (Kodak) for 20 min. Increasing concentrations (50-150 µg of protein/ml) of ox-LDL, native LDL (n-LDL), acetyl-LDL (Ac-LDL), and MDA-LDL were then added to the medium for 5 min at 37 °C in the presence of 5 mM arginine and 3 µM tetrahydrobiopterin (TB4). The incubation time was chosen on the basis of previous data showing that in these experimental conditions the ROS generation induced by ox-LDL increased rapidly in the first 5-6 min and then plateaued for longer ox-LDL incubations (15). Furthermore the short incubation time was chosen to avoid interferences derived from ox-LDL internalization. Samples were washed twice with phosphate-buffered saline containing bovine serum albumin and analyzed with 7000 cells per sample by flow cytometry (Coulter Electronics GmBH, Germany).

To test the response specificity, some radical scavengers such as vitamin C, trolox, and probucol (at a concentration of 5 µM; Sigma), anti-LOX-1 monoclonal antibody (mAb) (14), or comparable amounts of nonimmune mouse IgG (14) were incubated with BAECs, CHO-K1, and BLOX-1-CHO cells under the experimental conditions specified above.

To determine which oxidative systems contribute to the release of O&cjs1138;2 after ox-LDL exposure, BAECs were also preincubated with different amounts of L-N-monomethyl arginine (L-NMMA; 200 µM), L-N-arginine methyl ester (L-NAME; 200 µM), allopurinol (500 µM), aspirin (100 µM), and diphenyleneiodonium (DPI; 5 µM) for 30 min at 37 °C in presence of 5 mM arginine and 3 µM TB4. In our conditions, after incubation with n-LDL, ox-LDL, Ac-LDL, and MDA-LDL cell viability was always greater than 95%.

NO Measurement-- DAF-2 DA is a fluorescent indicator that enables the direct detection of NO under physiological conditions by flow cytometry (30). Confluent BAECs in 24-well plates were incubated in KRP (120 mM NaCl, 4.8 mM KCl, 0.54 mM CaCl2, 1.2 mM MgSO4, 11 mM glucose, 15.9 mM Na3PO3, pH 7.2) containing 10 µM DAF-2 DA for 10 min at 37 °C. Cells were then stimulated with 100 nM bradykinin and 150 mM thrombin for 5 min in presence of 5 mM arginine and 3 µM TB4. To verify whether the fluorescent signal obtained after the addition of DAF-2 DA was dependent on the presence of NO, L- and D-NMMA (200 µM) were preincubated with BAECs for 30 min before the addition of DAF-2 DA and NO agonists. Samples were then washed twice with phosphate-buffered saline containing bovine serum albumin and analyzed with 7000 cells per sample in flow cytometry (Coulter Electronics GmBH, Germany). In the same experimental conditions we also evaluated NO production by measuring levels of nitrite in the cell media by Griess reaction as described previously (31).

To evaluate the effect of ox-LDL on intracellular NO concentration, increasing amounts (50-150 µg of protein/ml) of ox-LDL, n-LDL, Ac-LDL, and MDA-LDL were incubated with BAECs for 0.5-15 min after the addition of DAF-2 DA and NO agonists, in the presence of 5 mM arginine and 3 µM TB4.

To verify whether the effect of ox-LDL on intracellular NO concentrations was dependent on ROS production and to test the response specificity, vitamin C, anti-LOX-1 mAb (14), or comparable amounts of nonimmune mouse IgG (14) were also used under the experimental conditions specified above.

Endothelial Nitric Oxide Synthase (eNOS) Activity Measurement-- The effect of ox-LDL on eNOS metabolism of 3H arginine to 3H citrulline was determined as described previously (32-34). The assay was performed under apparent Vmax conditions (32-34). Briefly BAECs lysates were suspended in cold lysis buffer (0.3 M sucrose, 10 mM HEPES, 1% Nonidet P-40, 0.1 mM EDTA, 1 mM dithiothreitol, 10 µg/ml leupeptin, 2 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, and 50 µM phenylmethylsulfonyl fluoride, pH 7.4) and vortexed. Cell lysates (150 to 250 µg of protein) were combined with NADPH (2 mM), CaCl2 (230 µM), TB4 (3 µM), and 3H-arginine (0.2 µCi, 10 µM) for 20 min at 37 °C. The assay volume was kept constant at 100 µl. To determine whether ox-LDL altered inducible NOS activity, the assay was repeated with EDTA (1.7 mM) replacing calcium in the assay buffers.

Statistical Analysis-- Statistical analysis was performed by analysis of variance and subsequently by post hoc analysis, using the SYSTAT program and statistical software manual (SYSTAT Inc., Evanston, IL) for Macintosh.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In our experimental conditions the incubations of BAECs with 10 µM DAF-2 DA for 10 min at 37 °C followed by stimulation with bradykinin or thrombin for 5 min generated a sharp increase of mean fluorescence intensity (MFI). This increase was dose-dependently suppressed by the NO synthase inhibitor L-NMMA whereas D-NMMA, the optical isomer of L-NMMA, was inactive. Fig. 1 shows the effect of 200 µM L-NMMA and D-NMMA on basal and stimulated NO production in BAECs.



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Fig. 1.   Effect of L- and D-NMMA on basal and stimulated intracellular NO concentration in BAECs. BAECs were preincubated with 200 µM L- and D-NMMA for 30 min at 37 °C before the addition of 10 µM DAF-2 DA for 10 min (Basal). Cells were then stimulated with 100 nM bradykinin and 150 mM thrombin for 5 min. Results are expressed as MFI and are the means ± S.D. of experiments performed in triplicate on six separate occasions. *, p < 0.001 versus control (no L- or D-NMMA).

Also the cumulative production of NO as evaluated by measuring levels of nitrite in the media was significantly increased after stimulation of BAECs with bradykinin or thrombin for 10 min at 37 °C (basal = 110 ± 7 pmol/well/h; after bradykinin = 370 ± 14 pmol/well/h, p < 0.001; after thrombin = 410 ± 12 pmol/well/h, p < 0.001).

The exposure of 1.7 mg of protein/ml of n-LDL to 5 µM Cu2+ for 18 at 37 °C resulted in a significant increase of thiobarbituric acid-reactive substances (11.9 ± 1.1 nmol/mg of LDL protein) compared with native LDL (0.24 ± 0.04 nmol/mg of LDL protein; p < 0.001).

The incubation of BAECs with increasing amounts of ox-LDL for 5 min in the presence of DAF-2 DA, dose-dependently reduced basal and bradykinin- or thrombin-induced intracellular NO formation (p < 0.001) (Fig. 2) whereas n-LDL did not (data not shown). Similarly Ac-LDL and MDA-LDL, even at the highest concentration (200 µg of protein/ml), had no effect (data not shown). The preincubation of BAECs with 200 µg of ox-LDL protein also significantly reduced the basal and stimulated levels of nitrite (basal from 102 ± 6 pmol/well/h to 44 ± 4 pmol/well/h, p < 0.01; after bradykinin from 352 ± 15 pmol/well/h to 121 ± 12 pmol/well/h, p < 0.01; after thrombin from 401 ± 14 pmol/well/h to 184 ± 9 pmol/well/h, p < 0.01). From the evaluation of the time course of nonstimulated and stimulated BAECs, it is evident that the effect of ox-LDL on intracellular NO production was already present after less than 60 s of incubation (Figs. 3, a-c).



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Fig. 2.   Effect of incubation of BAECs with increasing amounts of ox-LDL on basal and bradykinin- or thrombin-induced intracellular NO concentration. To evaluate the effect of ox-LDL on intracellular NO concentration, increasing amounts (50-200 µg of protein/ml) of ox-LDL were incubated with BAECs for 5 min after the addition of DAF-2 DA and NO agonists. Results are expressed as MFI and are the means ± S.D. of experiments performed in triplicate on six separate occasions. *, p < 0.001 versus time 0.



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Fig. 3.   Effect of ox-LDL on the time-course of NO concentration in basal (A) and stimulated (B and C) BAECs. BAECs were incubated for the indicated times with ox-LDL (100 µg of protein/ml) after the addition of DAF-2 DA and NO agonists. Results are expressed as MFI and are the means ± S.D. of experiments performed in triplicate on six separate occasions. *, p < 0.001 versus time 0.

The incubation of BAECs with ox-LDL for 5 min also induced a sharp and dose-dependent increase in intracellular concentration of ROS and O&cjs1138;2 (data not shown). The intracellular concentration of ROS and O&cjs1138;2 were slightly but not significantly increased by incubation with bradykinin and thrombin. In our experimental conditions, ox-LDL also triggered a strong and dose-dependent production of H2O2 after 5 min of incubation. With 50 µg of protein/ml of ox-LDL the mean value of H2O2 was 1.19 ± 0.3 nmol/106 cells. An almost identical response was seen for the production of extracellular O&cjs1138;2 as measured by cytochrome C with mean values of 5.9 ± 0.4 nmol/106 cells for 50 µg of protein/ml of ox-LDL.

To test the specificity of ROS and O&cjs1138;2 increase induced by ox-LDL in BAECs, we preincubated the cells with different antioxidants, known to work as radical scavengers. As shown in Fig. 4, trolox, probucol, and vitamin C significantly reduced the ox-LDL-induced ROS and O&cjs1138;2 production in BAECs (p < 0.001). Furthermore to verify whether the O&cjs1138;2 increase was dependent on ox-LDL binding to LOX-1 we preincubated BAECs, BLOX-1-CHO, and CHO-K1 cells with anti-LOX-1 mAb. For comparison the effect of vitamin C on ox-LDL-induced O&cjs1138;2 generation was also considered. As shown in Fig. 5, the O&cjs1138;2 concentration was markedly reduced in BAECs and BLOX-1-CHO cells preincubated with anti-LOX-1 mAb (p < 0.001), whereas control CHO-K1 cells were not affected.



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Fig. 4.   Effect of preincubation of BAECs with trolox, probucol, and vitamin C on ox-LDL-induced ROS and O&cjs1138;2 production. Trolox, probucol, and vitamin C (Vit. C) (5 µM) were preincubated with BAECs for 30 min; BAECs were then incubated for 5 min with ox-LDL (100 µg of protein/ml) after the addition of DAF-2 DA or HE. Results are expressed as percent variation (%) of the MFI induced by ox-LDL alone and are the means ± S.D. of experiments performed in triplicate on six separate occasions. *, p < 0.001 versus ox-LDL alone.



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Fig. 5.   Effect of vitamin C (Vit. C) and anti-LOX-1 mAb (LOX-1 Ab) on ox-LDL-induced variations of O&cjs1138;2 in BAECs, BLOX-1-CHO, and CHO cells. Vitamin C (5 µM), anti-LOX-1 mAb (30 µg/ml), and comparable amounts of nonimmune mouse IgG were preincubated with BAECs, BLOX-1-CHO, and CHO cells for 30 min. The cells were then incubated for 5 min with ox-LDL (100 µg of protein/ml) after the addition of HE. Results are expressed as MFI and are the means ± S.D. of experiments performed in triplicate on six separate occasions. *, p < 0.001 versus ox-LDL alone.

On the basis of the results described above, to test whether the reduction of intracellular NO concentration induced by ox-LDL was dependent on O&cjs1138;2 generation, we preincubated BAECs with vitamin C and anti-LOX-1 mAb. Fig. 6 shows that the preincubation of BAECs with vitamin C and anti-LOX-1 mAb significantly counteracted the effect of ox-LDL on basal and stimulated generation of NO (p < 0.001).



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Fig. 6.   Effect of vitamin C (Vit. C) and anti-LOX-1 mAb (LOX-1 Ab) on ox-LDL-induced variations of NO in basal and bradykinin- and thrombin-stimulated BAECs. Vitamin C (5 µM), anti-LOX-1 mAb (30 µg/ml), and comparable amounts of nonimmune mouse IgG were preincubated with BAECs for 30 min. BAECs were then incubated for 5 min with ox-LDL (100 µg of protein/ml) after the addition of DAF-2 DA and NO agonists. Results are expressed as MFI and are the means ± S.D. of experiments performed in triplicate on six separate occasions. *, p < 0.001 versus control; dagger , p < 0,01 versus ox-LDL.

The effect of ox-LDL on eNOS activity was examined by the 3H citrulline assay. ox-LDL did not significantly modify the ability of eNOS to metabolize L-arginine to L-citrulline (native LDL = 64.6 ± 9.4 pmol citrulline/mg protein/min; ox-LDL = 58.7 ± 8.9 pmol citrulline/mg protein/min, p = not significant). In presence of EDTA, the activity of inducible NOS was almost undetectable.

We also analyzed which oxidative systems may contribute to the release of O&cjs1138;2 after ox-LDL exposure (Fig. 7). We found that allopurinol did not affect O&cjs1138;2 whereas aspirin slightly but insignificantly reduced O&cjs1138;2 generation in BAECs. In contrast, L-NAME and L-NMMA but not D-NAME and D-NMMA significantly increased whereas DPI drastically reduced O&cjs1138;2 production in BAECs.



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Fig. 7.   Effect of L-NMMA, L-NAME, allopurinol, aspirin, and DPI on O&cjs1138;2 concentration induced by ox-LDL in BAECs. BAECs were preincubated with 200 µM L-NMMA, 200 µM L-NAME, 500 µM allopurinol, 100 µM aspirin, and 5 µM DPI for 30 min at 37 °C. After the addition of HE, BAECs were then incubated for 5 min with ox-LDL (100 µg of protein/ml). Results are expressed as MFI and are the means ± S.D. of experiments performed in triplicate on six separate occasions. *, p < 0.001 versus ox-LDL alone.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Using a novel fluorescence indicator, DAF-2 DA, for direct detection of NO (30), in this study we examined the relationship between the intracellular production of ROS and in particular of O&cjs1138;2 and the intracellular concentration of NO in culture of BAECs exposed to ox-LDL.

In our experimental conditions the incubations of BAECs with 10 µM DAF-2 DA for 10 min at 37 °C generated an increase in fluorescence intensity both in basal and agonist-stimulated cells, which was dose-dependently suppressed by the NO synthase inhibitor L-NMMA. These results are consistent with several lines of evidences suggesting that NO is generated under basal conditions by endothelial cells (35) and are in agreement with published results showing that 5-min exposure of BAECs to thrombin or bradykinin results in a sharp increase of NO (36). We found that ox-LDL, but not n-LDL or other forms of modified LDL, reduced in a dose-dependent fashion and very rapidly the intracellular NO concentration in basal and stimulated endothelial cells.

Our results agree with a series of studies addressing the effects of ox-LDL on arterial rings (37, 38) and on cultured endothelial cells (39). These effects have been seen to occur when vascular segments or cultured cells are placed in contact with LDL for long periods suggesting inhibition of NO synthesis by ox-LDL. Even if there is no agreement regarding interpretation of this phenomenon (38-40), in our experimental conditions ox-LDL did not significantly alter the ability of eNOS to metabolize L-arginine to L-citrulline. Because the conversion of 3H arginine into 3H citrulline, under apparent Vmax conditions (32-34), is a measure of eNOS levels, the results of this study show that ox-LDL did not alter, at least quantitatively, the ability to produce NO.

Interestingly and in agreement with very recent data published by our group (15), the results of this study also demonstrate that the rapid decrease in NO induced by ox-LDL was parallelled by a specular fast increase in ROS and O&cjs1138;2 formation. The increased cellular production of ROS and O&cjs1138;2 in particular was prevented by preincubating BAECs with different antioxidants known to work as radical scavengers. These data confirm that the incubation of ox-LDL with BAECs is associated with an increased intracellular production of ROS and O&cjs1138;2 (15). Furthermore the fact that in this study the generation of ROS was prevented by anti-LOX-1 mAb and the fact that the formation of ROS was reported to persist for longer ox-LDL incubations (15), strongly support that the ox-LDL ligation to LOX-1 plays a role in intracellular ROS generation (15).

The decrease of intracellular NO concentration was prevented by preincubating BAECs with different antioxidants known to work as radical scavengers and with anti-LOX-1 mAb. The data support the conclusion that the incubation of ox-LDL with BAECs is associated with a receptor-dependent, abnormally increased intracellular production of ROS and in particular of O&cjs1138;2. NO decrease therefore may be secondary to O&cjs1138;2 formation, which is known to inactivate NO in a chemical reaction during which the cytotoxic radical peroxynitrite is formed (9, 10). Furthermore, because in our experimental conditions the effect of ox-LDL on intracellular NO concentration was receptor-mediated and consequently very fast, the signal of NO reduction being clearly visible even after 30 s, it is unlikely that the effect on NO is secondary to its reaction with intracellularly internalized ox-LDL lipid peroxyl radicals.

There are many enzymatic sources for ROS in almost all cell types (41), and several findings have indicated an increase in ROS upon receptor ligation (42-46). As for the potential sources of ROS induced by the binding of ox-LDL to LOX-1, we found that allopurinol and aspirin did not significantly affect O&cjs1138;2 generation in BAECs. Because the levels of allopurinol and aspirin used in this study have already been shown to completely block xantine oxidase activity (47) and arachidonic acid metabolism by cycloxygenase (48), the results of this study indicate that the generation of O&cjs1138;2 induced by the ligation of ox-LDL to LOX-1 is not related to the activity of these enzymes. Furthermore the fact that L-NAME and L-NMMA did not reduce O&cjs1138;2 generation by BAECs reasonably excludes that, in our experimental conditions, eNOS is involved in O&cjs1138;2 generation. Our results agree with the findings of Pou et al. (49) who found that L-NMMA blocked neuronal brain NOS O&cjs1138;2 production only under uncoupling conditions. Because we worked in excess of L-arginine and TB4, our results are also consistent with the results of Vergnani et al. (50) who showed that L-arginine drastically reduced the ox-LDL-induced O&cjs1138;2 generation by endothelial cell under uncoupling conditions. Our findings that L-NAME and L-NMMA increased and not decreased intracellular O&cjs1138;2 concentration further support the conclusion that in our study eNOS is not involved in ox-LDL-induced O&cjs1138;2 generation. Even if this phenomenon is not completely clear, one hypothesis could be that in the presence of eNOS inhibitors, less O&cjs1138;2 is quenched by the reduced intracellular NO.

Finally the results of this study clearly show that DPI drastically reduced O&cjs1138;2 generation in BAECs stimulated by ox-LDL. Of course, on the basis of this result we cannot draw any conclusive assumption on which oxidative system contributes to the release of O&cjs1138;2 after ox-LDL exposure. DPI, a selective inhibitor of NADPH oxidase (51), also inhibits mitochondrial O&cjs1138;2 production by NADH ubiquinone oxidoreductase (complex I) (52) and eNOS (53). However, because in our experimental conditions it is unlikely that eNOS is involved in O&cjs1138;2 generation, another conclusion of this study is that the generation of O&cjs1138;2 after ox-LDL exposure may be related to the increased activity of NADPH oxidase and/or mitochondrial complex 1.

The reduction in intracellular NO concentration as a result of O&cjs1138;2 generation may also have implications in vivo. In fact, it has been recognized for a long time that atherosclerotic blood vessels are very susceptible to the development of vasospasm in vivo (4, 54-56), and the current weight of evidence suggests that impaired endothelium-dependent vasodilatation is the predominant mechanism underlying inappropriate constriction leading to ischemic manifestations (57-59).

Increased oxidative stress within the vascular wall facilitates oxidation of LDL (60). The ROS produced by the ligation of ox-LDL to LOX-1 could facilitate the oxidation of native LDL or partially oxidized LDL, which in turn could up-regulate LOX-1 expression (61) and contribute to further O&cjs1138;2 generation, which could finally inactivate NO in a chemical reaction during which peroxynitrite is formed. The phenomenon could be amplified in pathological situations characterized by higher plasma concentration of LDL like hypercholesterolemia. Of course this conclusion is limited by the fact that in this study we measured only the rapid response of ROS and O&cjs1138;2 induced by ox-LDL. However, because ROS generation was reported to persist for longer ox-LDL incubations (15), we are tempted to speculate that this oxidative stress would continue to inactivate NO. In conclusion the results of this study show that one of the pathophysiological consequences of ox-LDL binding to LOX-1 may be the inactivation of NO through an increased cellular production of O&cjs1138;2.


    FOOTNOTES

* This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Ministry of Health, Labour and Welfare of Japan; the Organization for Pharmaceutical Safety and Research; Takeda Science Foundation; and DNO Medical Research Foundation.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.

Dagger To whom correspondence should be addressed: Dipartimento di Scienze Biomediche e Chirurgiche, c/o Medicina D, Ospedale Policlinico, Università di Verona, 37134 Verona, Italy. Tel.: 39-045-8074806; Fax: 39-045-583041; E-mail: comina@medicinad.univr.it.

Published, JBC Papers in Press, January 24, 2001, DOI 10.1074/jbc.M010612200


    ABBREVIATIONS

The abbreviations used are: NO, nitric oxide; O&cjs1138;2, superoxide; ox-LDL, oxidized low density lipoprotein; LOX-1, lectin-like ox-LDL receptor-1; BAEC(s), bovine aortic endothelial cell(s); ROS, reactive oxygen species; MDA-LDL, malondialdehyde-modified LDL; CHO, Chinese hamster ovary; BLOX-1, bovine LOX-1; HE, hydroethidine; n-LDL, native LDL; Ac-LDL, acetyl-LDL; TB4, tetrahydrobiopterin; L-NMMA, L-N-monomethyl arginine; L-NAME, L-N-arginine methyl ester; DPI, diphenyleneiodonium; eNOS, endothelial nitric oxide synthase; MFI, mean fluorescence intensity; D-NMMA, D-N-monomethyl arginine; DAF-2 DA, 4,5 diaminofluorescein diacetate.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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