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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

2001; Elsevier BV; Volume: 276; Issue: 17 Linguagem: Inglês

10.1074/jbc.m010612200

ISSN

1083-351X

Autores

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

Tópico(s)

Antioxidant Activity and Oxidative Stress

Resumo

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⨪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⨪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⨪2 generation (p < 0.001). In the presence of radical scavengers and anti-LOX-1 monoclonal antibody, O⨪2formation 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 metabolizel-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⨪2. 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⨪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⨪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⨪2 generation (p < 0.001). In the presence of radical scavengers and anti-LOX-1 monoclonal antibody, O⨪2formation 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 metabolizel-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⨪2. Endothelium-dependent relaxation is impaired in animals with atherosclerosis (1Guerra Jr., R. Brotherthon A.F.A. Goodwin P.J. Clark C.R. Armstrong M.L. Harrison D.G. Blood Vessels. 1989; 26: 300-314PubMed Google Scholar, 2Girerd X.J. Hirsh A.T. Cooke J.P. Dzau V.J. Creager M.A. Circ. Res. 1990; 67: 1301-1308Crossref PubMed Scopus (242) Google Scholar, 3Drexler H. Zeiher A.M. Meinzer K. Just H. Lancet. 1991; 338: 1546-1550Abstract PubMed Scopus (719) Google Scholar), which has been linked to a decreased production and/or biological activity of endothelium-derived nitric oxide (NO) 1The abbreviations used are: NOnitric oxideO⨪2superoxideox-LDLoxidized low density lipoproteinLOX-1lectin-like ox-LDL receptor-1BAEC(s)bovine aortic endothelial cell(s)ROSreactive oxygen speciesMDA-LDLmalondialdehyde-modified LDLCHOChinese hamster ovaryBLOX-1bovine LOX-1HEhydroethidinen-LDLnative LDLAc-LDLacetyl-LDLTB4tetrahydrobiopterinl-NMMAl-N-monomethyl argininel-NAMEl-N-arginine methyl esterDPIdiphenyleneiodoniumeNOSendothelial nitric oxide synthaseMFImean fluorescence intensityd-NMMAd-N-monomethyl arginineDAF-2 DA4,5 diaminofluorescein diacetate (4Cooke J.P. Andon N.A. Girerd X.J. Hirsh A.T. Creager M.A. Circulation. 1991; 83: 1057-1062Crossref PubMed Scopus (313) Google Scholar, 5Greene B. Pacitti A.J. Souba W.W. Am. J. Physiol. 1993; 264: 351-356PubMed Google Scholar). Oxidative inactivation of NO is regarded as an important cause of its decreased biological activity (6Boger R.H. Bode-Boger S.M. Frolich J.C. Atherosclerosis. 1996; 127: 1-11Abstract Full Text PDF PubMed Scopus (161) Google Scholar). The vascular release of superoxide (O⨪2) radicals is sharply increased in atherosclerotic arteries (7Mugge A. Brandes R. Boger R.H. Dwenger A. Bade-Boger S. Kienke S. Frolich J.C. Lichtlen P.R. J. Cardiovasc. Pharmacol. 1994; 24: 994-998Crossref PubMed Scopus (128) Google Scholar, 8Ohara Y. Peterson T.E. Harrison D.G. J. Clin. Invest. 1993; 91: 2546-2551Crossref PubMed Scopus (1656) Google Scholar), and O⨪2 is known to inactivate NO in a chemical reaction during which the cytotoxic radical peroxynitrite is formed (9Ignarro R.J. Annu. Rev. Pharmacol. Toxicol. 1990; 30: 535-560Crossref PubMed Scopus (1224) Google Scholar,10Huie R.E. Padmaja S. Free Radic. Res. 1993; 18: 195-199Crossref Scopus (2028) Google Scholar). The presence of peroxynitrite-derived nitrotyrosines has recently been demonstrated in human atherosclerotic lesions (11Beckman J.S. Ye Y.Z. Anderson P.G. Chen J. Accavitti M.A. Tarpey M.M. White C.R. Biol. Chem. Hoppe-Seyler. 1994; 375: 81-88Crossref PubMed Scopus (1076) Google Scholar). nitric oxide superoxide oxidized low density lipoprotein lectin-like ox-LDL receptor-1 bovine aortic endothelial cell(s) reactive oxygen species malondialdehyde-modified LDL Chinese hamster ovary bovine LOX-1 hydroethidine native LDL acetyl-LDL tetrahydrobiopterin l-N-monomethyl arginine l-N-arginine methyl ester diphenyleneiodonium endothelial nitric oxide synthase mean fluorescence intensity d-N-monomethyl arginine 4,5 diaminofluorescein diacetate 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 (12Witztum J.L. Lancet. 1994; 344: 793-795Abstract PubMed Scopus (1261) Google Scholar). 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 (13Noll G. Luscher T.F. Thromb. Res. 1994; 74 Suppl. 1: S45-S54Abstract Full Text PDF PubMed Scopus (30) Google Scholar). 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) (14Sawamura T. Kume N. Aoyama T. Moriwaki H. Hoshikawa H. Alba Y. Tnaka T. Miwa S. Katsura Y. Kita T. Masaki T. Nature. 1997; 386: 73-77Crossref PubMed Scopus (1176) Google Scholar). It has been suggested that ox-LDL uptake through this receptor may be involved in endothelial activation or dysfunction in atherogenesis (14Sawamura T. Kume N. Aoyama T. Moriwaki H. Hoshikawa H. Alba Y. Tnaka T. Miwa S. Katsura Y. Kita T. Masaki T. Nature. 1997; 386: 73-77Crossref PubMed Scopus (1176) Google Scholar). 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 (15Cominacini L. Fratta Pasini A. Garbin U. Davoli A. Tosetti L. Campagnola M. Rigoni A. Pastorino A.M. Lo Cascio V. Sawamura T. J. Biol. Chem. 2000; 275: 12633-12638Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar). In this report we investigated the relationship between the intracellular production of ROS and in particular of O⨪2and the intracellular concentration of NO in cultures of BAECs exposed to ox-LDL. 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 (16Havel R.J. Eder M.A. Bragdon J.M. J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6498) Google Scholar) containing 1 mg/ml EDTA. Cu2+-modified LDL (1.7 mg of protein/ml) was prepared by exposure of LDL to 5 βm CuS04 for 18 h at 37 °C as described previously (17Cominacini L. Garbin U. Davoli A. Micciolo R. Bosello O. Gaviraghi G. J. Lipid Res. 1991; 32: 349-358Abstract Full Text PDF PubMed Google Scholar, 18Cominacini L. Garbin U. De Santis A. Campagnola M. Davoli A. Fratta Pasini A. Faccini G. Pasqualini E. Bertozzo L. Micciolo R. Pastorino A.M. Lo Cascio V. J. Lipid Med. 1996; 13: 19-33Google Scholar). The extent of LDL oxidation was determined by thiobarbituric acid-reactive substances as reported (18Cominacini L. Garbin U. De Santis A. Campagnola M. Davoli A. Fratta Pasini A. Faccini G. Pasqualini E. Bertozzo L. Micciolo R. Pastorino A.M. Lo Cascio V. J. Lipid Med. 1996; 13: 19-33Google Scholar). Protein was measured by the Pierce BCA protein assay reagent (19Smith P.K. Krohn R.I. Hermanson G.T. Mallia A. Gartner F.H. Provenzano A.C. Anal. Biochem. 1985; 105: 293-299Google Scholar). Malondialdehyde-modified LDL (MDA-LDL) was prepared according to a previously described method (20Haberland M.E. Fogelman A.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 82: 2693-2697Crossref Scopus (86) Google Scholar, 21Habeeb A.F. Anal. Biochem. 1966; 14: 328-336Crossref PubMed Scopus (1936) Google Scholar). Acetylation of LDL was achieved by repeated additions of acetic anhydride (22Sparrow C.P. Parthasarathy S. Steinberg D. J. Biol. Chem. 1989; 264: 2599-2604Abstract Full Text PDF PubMed Google Scholar). BAECs were isolated and cultured as described previously (23Murase T. Kume N. Korenaga R. Ando J. Sawamura T. Masaki T. Kita T. Circ. Res. 1998; 83: 328-333Crossref PubMed Scopus (188) Google Scholar). 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) (14Sawamura T. Kume N. Aoyama T. Moriwaki H. Hoshikawa H. Alba Y. Tnaka T. Miwa S. Katsura Y. Kita T. Masaki T. Nature. 1997; 386: 73-77Crossref PubMed Scopus (1176) Google Scholar) were cultured as described previously (23Murase T. Kume N. Korenaga R. Ando J. Sawamura T. Masaki T. Kita T. Circ. Res. 1998; 83: 328-333Crossref PubMed Scopus (188) Google Scholar). Cell survival was monitored according to the method of Landegren (24Landegren U. J. Immunol. Methods. 1984; 67: 379-385Crossref PubMed Scopus (603) Google Scholar). Intracellular ROS production was monitored by following the oxidation of 2′,7′-dichlorofluorescin diacetate in flow cytometry as described by Royall (25Royall J.A. Ischiropoulos H. Arch. Biochem. Biophys. 1993; 302: 348-355Crossref PubMed Scopus (1050) Google Scholar) and slightly modified by Zulueta (26Zulueta J.J. Sawhney R. Feng S.Y. Cote C.C. Hassoun P.M. Am. J. Physiol. 1997; 272: 897-902Crossref PubMed Google Scholar). Intracellular O⨪2 generation was detected using a previously established flow cytometry technique based on the O⨪2-induced conversion of the oxidant-sensitive dye, hydroethidine (HE) to ethidium (27Rothe G. Valet G. J. Leukocyte Biol. 1990; 47: 440-446Crossref PubMed Scopus (783) Google Scholar). 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 (28Zhou M. Diwu Z. Panhuk-Voloshina N. Haugland R.P. Anal. Biochem. 1997; 15: 162-168Crossref Scopus (1126) Google Scholar) and, that of O⨪2 was determined photometrically by reduction of cytochrome c(29McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar). Confluent BAECs in 24-well plates were incubated in Dulbecco's modified Eagle's medium (Sigma) containing 10% fetal bovine serum, 10 βm 2′,7′-dichlorofluorescin diacetate (Eastman Kodak Co., Rochester, NY), or 1 βm HE (Kodak) for 20 min. Increasing concentrations (50–150 βg 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 mmarginine and 3 βm 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 (15Cominacini L. Fratta Pasini A. Garbin U. Davoli A. Tosetti L. Campagnola M. Rigoni A. Pastorino A.M. Lo Cascio V. Sawamura T. J. Biol. Chem. 2000; 275: 12633-12638Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar). 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 βm; Sigma), anti-LOX-1 monoclonal antibody (mAb) (14Sawamura T. Kume N. Aoyama T. Moriwaki H. Hoshikawa H. Alba Y. Tnaka T. Miwa S. Katsura Y. Kita T. Masaki T. Nature. 1997; 386: 73-77Crossref PubMed Scopus (1176) Google Scholar), or comparable amounts of nonimmune mouse IgG (14Sawamura T. Kume N. Aoyama T. Moriwaki H. Hoshikawa H. Alba Y. Tnaka T. Miwa S. Katsura Y. Kita T. Masaki T. Nature. 1997; 386: 73-77Crossref PubMed Scopus (1176) Google Scholar) 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⨪2 after ox-LDL exposure, BAECs were also preincubated with different amounts of l-N-monomethyl arginine (l-NMMA; 200 βm),l-N-arginine methyl ester (l-NAME; 200 βm), allopurinol (500 βm), aspirin (100 βm), and diphenyleneiodonium (DPI; 5 βm) for 30 min at 37 °C in presence of 5 mm arginine and 3 βm TB4. In our conditions, after incubation with n-LDL, ox-LDL, Ac-LDL, and MDA-LDL cell viability was always greater than 95%. DAF-2 DA is a fluorescent indicator that enables the direct detection of NO under physiological conditions by flow cytometry (30Kojima H. Nakatsubo N. Kikuchi K. Urano Y. Higuchi T. Tanaka J. Kudo Y. Nagano N. NeuroReport. 1998; 9: 3345-3348Crossref PubMed Scopus (189) Google Scholar). 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 mmNa3PO3, pH 7.2) containing 10 βmDAF-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 βm TB4. To verify whether the fluorescent signal obtained after the addition of DAF-2 DA was dependent on the presence of NO, l- andd-NMMA (200 βm) 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 (31Schneider N.R. Yeary N.A. Am. J. Vet. Res. 1973; 34: 133-135PubMed Google Scholar). To evaluate the effect of ox-LDL on intracellular NO concentration, increasing amounts (50–150 βg 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 βm 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 (14Sawamura T. Kume N. Aoyama T. Moriwaki H. Hoshikawa H. Alba Y. Tnaka T. Miwa S. Katsura Y. Kita T. Masaki T. Nature. 1997; 386: 73-77Crossref PubMed Scopus (1176) Google Scholar), or comparable amounts of nonimmune mouse IgG (14Sawamura T. Kume N. Aoyama T. Moriwaki H. Hoshikawa H. Alba Y. Tnaka T. Miwa S. Katsura Y. Kita T. Masaki T. Nature. 1997; 386: 73-77Crossref PubMed Scopus (1176) Google Scholar) were also used under the experimental conditions specified above. The effect of ox-LDL on eNOS metabolism of3H arginine to 3H citrulline was determined as described previously (32Sessa W.C. Harrison J.K. Luthin D.R. Pollock J.S. Lynch K.R. Hypertension. 1993; 21: 934-938Crossref PubMed Scopus (82) Google Scholar, 33Sessa W.C. Barber C.M. Lynch K.R. Circ. Res. 1993; 72: 921-924Crossref PubMed Scopus (132) Google Scholar, 34Rosenkranz-Weiss P. Sessa W.C. Milstien S. Kaufman S. Watson J.A. Pober J.S. J. Clin. Invest. 1994; 93: 2236-2243Crossref PubMed Scopus (344) Google Scholar). The assay was performed under apparent Vmax conditions (32Sessa W.C. Harrison J.K. Luthin D.R. Pollock J.S. Lynch K.R. Hypertension. 1993; 21: 934-938Crossref PubMed Scopus (82) Google Scholar, 33Sessa W.C. Barber C.M. Lynch K.R. Circ. Res. 1993; 72: 921-924Crossref PubMed Scopus (132) Google Scholar, 34Rosenkranz-Weiss P. Sessa W.C. Milstien S. Kaufman S. Watson J.A. Pober J.S. J. Clin. Invest. 1994; 93: 2236-2243Crossref PubMed Scopus (344) Google Scholar). Briefly BAECs lysates were suspended in cold lysis buffer (0.3 msucrose, 10 mm HEPES, 1% Nonidet P-40, 0.1 mmEDTA, 1 mm dithiothreitol, 10 βg/ml leupeptin, 2 βg/ml aprotinin, 10 βg/ml soybean trypsin inhibitor, and 50 βm phenylmethylsulfonyl fluoride, pH 7.4) and vortexed. Cell lysates (150 to 250 βg of protein) were combined with NADPH (2 mm), CaCl2 (230 βm), TB4 (3 βm), and 3H-arginine (0.2 βCi, 10 βm) for 20 min at 37 °C. The assay volume was kept constant at 100 βl. 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 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. In our experimental conditions the incubations of BAECs with 10 βm 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 inhibitorl-NMMA whereas d-NMMA, the optical isomer ofl-NMMA, was inactive. Fig. 1shows the effect of 200 βml-NMMA andd-NMMA on basal and stimulated NO production in BAECs. 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 βmCu2+ 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 βg of protein/ml), had no effect (data not shown). The preincubation of BAECs with 200 βg 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).Figure 3Effect 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 βg 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 < 0.001versus time 0.View Large Image Figure ViewerDownload (PPT) 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⨪2 (data not shown). The intracellular concentration of ROS and O⨪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 H2O2after 5 min of incubation. With 50 βg 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⨪2 as measured by cytochrome C with mean values of 5.9 ± 0.4 nmol/106 cells for 50 βg of protein/ml of ox-LDL. To test the specificity of ROS and O⨪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⨪2 production in BAECs (p < 0.001). Furthermore to verify whether the O⨪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⨪2generation was also considered. As shown in Fig.5, the O⨪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.Figure 5Effect of vitamin C (Vit. C) and anti-LOX-1 mAb ( LOX-1 Ab) on ox-LDL-induced variations of O⨪2 in BAECs, BLOX-1-CHO, and CHO cells.Vitamin C (5 βm), anti-LOX-1 mAb (30 βg/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 βg 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.View Large Image Figure ViewerDownload (PPT) 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⨪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). The effect of ox-LDL on eNOS activity was examined by the3H citrulline assay. ox-LDL did not significantly modify the ability of eNOS to metabolize l-arginine tol-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⨪2 after ox-LDL exposure (Fig.7). We found that allopurinol did not affect O⨪2 whereas aspirin slightly but insignificantly reduced O⨪2 generation in BAECs. In contrast, l-NAME andl-NMMA but not d-NAME and d-NMMA significantly increased whereas DPI drastically reduced O⨪2production in BAECs. Using a novel fluorescence indicator, DAF-2 DA, for direct detection of NO (30Kojima H. Nakatsubo N. Kikuchi K. Urano Y. Higuchi T. Tanaka J. Kudo Y. Nagano N. NeuroReport. 1998; 9: 3345-3348Crossref PubMed Scopus (189) Google Scholar), in this study we examined the relationship between the intracellular production of ROS and in particular of O⨪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 βm 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 (35Palmer R.M.J. Ashton D.S. Moncada S. 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The increased cellular production of ROS and O⨪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⨪2 (15Cominacini L. Fratta Pasini A. Garbin U. Davoli A. Tosetti L. Campagnola M. Rigoni A. Pastorino A.M. Lo Cascio V. Sawamura T. J. Biol. Chem. 2000; 275: 12633-12638Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar). 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 (15Cominacini L. Fratta Pasini A. Garbin U. Davoli A. Tosetti L. Campagnola M. Rigoni A. Pastorino A.M. Lo Cascio V. Sawamura T. J. Biol. 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Yeung A.C. Vekstein V.I. Selwyn A.P. Ganz P. Circulation. 1990; 81: 491-497Crossref PubMed Scopus (1091) Google Scholar, 58Vanhoutte P.M. Shimokawa H. Circulation. 1989; 80: 1-9Crossref PubMed Scopus (284) Google Scholar, 59Sneddon J.M. Vane J.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2800-2804Crossref PubMed Scopus (241) Google Scholar). Increased oxidative stress within the vascular wall facilitates oxidation of LDL (60Steinbrecher U.P. Biochim. Biophys. Acta. 1988; 959: 20-30Crossref PubMed Scopus (202) Google Scholar). 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 (61Kume N. Murase T. Moriwaki H. Aoyama T. Sawamura T. Masaki T. Kita T. Circ. Res. 1998; 81: 322-327Crossref Scopus (278) Google Scholar) and contribute to further O⨪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⨪2 induced by ox-LDL. However, because ROS generation was reported to persist for longer ox-LDL incubations (15Cominacini L. Fratta Pasini A. Garbin U. Davoli A. Tosetti L. Campagnola M. Rigoni A. Pastorino A.M. Lo Cascio V. Sawamura T. J. Biol. Chem. 2000; 275: 12633-12638Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar), 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⨪2.

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