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Circulation. 1997;95:723-731

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(Circulation. 1997;95:723-731.)
© 1997 American Heart Association, Inc.

Articles

Inhibitors of Arterial Relaxation Among Components of Human Oxidized Low-Density Lipoproteins

Cholesterol Derivatives Oxidized in Position 7 Are Potent Inhibitors of Endothelium-Dependent Relaxation

Valerie Deckert, PhD; Laurence Persegol, PhD; Laurence Viens, MS; Gerard Lizard, PhD; Anne Athias, MS; Christian Lallemant, MD; Philippe Gambert, MD, PhD; Laurent Lagrost, PhD

the Laboratoire de Biochimie des Lipoproteines, INSERM CJF 93-10, Faculte de Medecine, Dijon, France.

Correspondence to Laurent Lagrost, Laboratoire de Biochimie Medicale, Hopital du Bocage, 21034 Dijon, France.


*    Abstract
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 *Abstract
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 down arrowResults
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Background Oxidized low-density lipoproteins (LDLs) are known to impair arterial relaxation. The aim of the present study was to identify the components of oxidized LDL that may account for inhibition of endothelium-dependent relaxation.

Methods and Results LDLs from 12 healthy subjects were either maintained at 4°C (native LDL) or incubated at 37°C in the presence of copper sulfate (oxidized LDL). Unlike pretreatment with native LDL, pretreatment with oxidized LDL reduced significantly the acetylcholine-mediated relaxation of rabbit aortic segments compared with control segments incubated in Krebs' buffer (maximal relaxation [Emax], 72.0±6.7% versus 94.1±0.8%, respectively, P<.01; negative logarithm of the concentration required to produce a half-maximal relaxing effect [pD2], 6.6±0.1 versus 7.2±0.1, respectively, P<.001). The absolute difference between Emax values obtained with oxidized and native LDL ({Delta}Emax) correlated significantly with the formation of 7-ketocholesterol, 7{alpha}-hydroxycholesterol, and 7ß-hydroxycholesterol. In contrast, {Delta}Emax did not correlate with the amount of lipoperoxides or lysophosphatidylcholine formed, and the difference of pD2 values measured with oxidized and native LDL ({Delta}pD2) did not correlate significantly with any of the oxidation-derived LDL compounds. When added individually, 7-ketocholesterol and 7ß-hydroxycholesterol reduced Emax values but not pD2 values in a time- and concentration-dependent manner.

Conclusions Cholesterol derivatives in oxidized LDL can reduce maximal arterial relaxation through a specific effect on vascular endothelial cells.


Key Words: lipoproteins • cholesterol • endothelium • acetylcholine • vessels


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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It has been well established that hyperlipidemia and atherosclerosis are associated with abnormalities of the vascular function characterized both by an increase in the response to specific vasoconstrictor agents1 2 3 4 5 and by a marked attenuation of endothelium-dependent relaxation.4 5 6 7 These alterations of vascular reactivity occur at an early stage of the atherosclerotic process,3 7 8 9 10 and they may predispose arteries to the development of vasospasm. Whereas some studies suggested that native LDL might inhibit endothelium-dependent relaxation,11 12 possibly through the inactivation of NO by lipoprotein particles,12 LDLs were demonstrated by several groups13 14 15 16 to impair arterial relaxation only in their oxidized form. In support of the latter view, Plane et al17 found that the high susceptibility of LDLs to undergo oxidative transformations is associated with marked inhibition of vascular relaxation. Although generation of LPC during the oxidative process of LDL has been proposed to account at least in part for the ability of oxidized LDLs to impair the endothelium-dependent relaxation of rabbit aorta,14 16 18 LPC did not mimic the inhibitory effect of oxidized LDL in porcine coronary arteries,15 and the search for a correlation of the lysolecithin content of oxidized LDLs with their inhibitory potential on rabbit aortic rings led to inconsistent results.17 18 These observations suggest, therefore, that in addition to LPC, other components of oxidized LDL, such as cholesterol oxides or lipoperoxides, might be involved in impairment of endothelium-dependent arterial relaxation.

In the present study, LDLs isolated from several normolipidemic human plasma samples were oxidized in vitro, and their composition as well as their ability to inhibit the ACh-mediated endothelium-dependent relaxation of isolated rabbit aorta were studied. The relaxation of the rabbit aorta in response to ACh is mediated by the endothelium-derived relaxing factor–NO system.19 20 In a complementary approach, the effects of various isolated compounds generated during LDL oxidation on endothelium-dependent relaxation were addressed.


*    Methods
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 up arrowIntroduction
 *Methods
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Preparation of LDLs
LDLs were isolated by sequential ultracentrifugation from fresh citrated plasma drawn from 12 normolipidemic donors (6 females and 6 males aged 36±3 years; triglycerides, 136±16 mg/dL; total cholesterol, 202±8 mg/dL; HDL cholesterol, 57±4 mg/dL; and apo B, 99±4 mg/dL). Densities were adjusted by the addition of KBr. The 1.019<d<1.063-g/mL fraction was prepared from 150 mL plasma in a 70.1 rotor on an L7 ultracentrifuge (Beckman) with one 15-hour, 45 000-rpm run at the lowest density and one 24-hour, 45 000-rpm run at the highest density. The LDL fractions were dialyzed overnight against a 10 mmol/L Tris, 150 mmol/L NaCl, pH 7.4 buffer (TBS). LDL preparations from different normolipidemic donors were not pooled.

Oxidative Modification of LDL
The oxidative modification of LDL was performed by incubating freshly prepared LDLs adjusted to 1.2 g protein/L in TBS with a copper sulfate solution (final concentration, 5 µmol/L) for 24 hours at 37°C. At the end of the oxidation period, oxidation was stopped by the addition of EDTA (final concentration, 200 µmol/L) and BHT (final concentration, 20 µmol/L). In parallel, native LDLs were obtained by supplementing freshly prepared LDLs (1.2 g protein/L) with EDTA (final concentration, 200 µmol/L) and BHT (final concentration, 20 µmol/L ). The native LDL preparations were maintained for 24 hours at 4°C. Finally, after the 24-hour period, both native and oxidized LDL preparations were dialyzed overnight against Krebs' buffer with the following composition (in mmol/L): NaCl 119, KCl 4.7, KH2PO4 1.18, MgSO4 1.17, CaCl2 2.5, EDTA 0.027, glucose 11, and NaHCO3 25.

The ability of various LDLs to alter vascular reactivity was determined by the use of nonfrozen LDL fractions within 2 days after their preparation. For biochemical measurements, aliquots of LDL preparations were stored for a few weeks at -80°C under nitrogen. LDL preparations were not sterilized before storage.

Assay of Lipoperoxides
Lipoperoxides were measured in both native and oxidized LDLs by the spectrophotometric method described by El-Saadani et al,21 adapted on a COBAS-BIO centrifugal analyzer (Roche).

Assay of Cholesterol Oxides
Cholesterol oxides were analyzed by capillary gas chromatography22 on a Hewlett-Packard 12.5-m-long fused silica, cross-linked methylsilicone column with the use of a Hewlett-Packard 5890 gas chromatograph attached to a 5971A mass detector (Hewlett-Packard). Concentrations of cholesterol and cholesterol oxides were determined from the ratio of the peak area corresponding to one given molecule to the peak area corresponding to the internal standard (epicoprostanol).

Assay of LPC
LDL phospholipids were assayed by HPLC23 with the use of a Gold HPLC System (Beckman) on a 220x4.6-mm Spheri-5 silice 5-µm column (Brownlee) that was connected to a light-scattering detector (model DDL21; Cunow). Phosphatidyl-N,N-dimethylethanolamine-dipalmitoyl was added as an internal standard. LPC contents were determined from the ratio of the peak area of the sample to the peak area of the internal standard. As checked by adding known amounts of purified LPC to LDLs before lipid extraction, the recovery of LPC constantly exceeded 80% of total.

Determination of Fatty Acid Composition of LDLs
Total fatty acids in native and oxidized LDLs were analyzed by capillary gas chromatography24 on a Hewlett-Packard 5890 gas chromatograph attached to a 5971A mass detector (Hewlett-Packard). Heptadecanoic acid (17:0) was added as an internal standard, and the fatty acid contents were determined from the ratio of the peak area of the sample to the peak area of the internal standard.

Analysis of Antioxidants in LDLs
{alpha}-Tocopherol, {gamma}-tocopherol, {alpha}-carotene, and ß-carotene were assayed by use of HPLC according to the general method described by Miller and Yang.25 The chromatographic analysis was performed by use of a Gold HPLC System (Beckman) on a 220x4.6-mm Spheri-5 RP 18 column (Brownlee) that was connected to a diode array detector (model 168; Beckman). {delta}-Tocopherol was added to each sample as an internal standard before the extraction, and the antioxidant contents were determined from the ratio of the peak area of the sample to the peak area of the internal standard.

Preparation of Blood Vessels
Fifty-five New Zealand White rabbits of either sex weighing 2.8 to 3.3 kg were killed by an overdose of pentobarbital sodium via the marginal ear vein. The descending aorta was rapidly removed and transferred into a Krebs' solution bubbled with 95% O2 plus 5% CO2. The aortas were cut into 3-mm rings and suspended horizontally between two wire hooks in 20-mL jacketed organ baths containing oxygenated Krebs' solution (composition in mmol/L: NaCl 119, KCl 4.7, KH2PO4 1.18, MgSO4 1.17, CaCl2 2.5, EDTA 0.027, glucose 11, and NaHCO3 25) maintained at 37°C. One hook was connected to a force transducer (model UF1, Pioden Ltd) and the other was fixed to the support. Changes in isometric tension were monitored continuously on a Mac Lab 8 system (AD Instruments Ltd). The resting tension of the rings was set to {approx}2 g and was similar to the resting tension used in many previous studies conducted with rabbit aortic rings.11 16 26 After a 30-minute equilibration period, the contractile response to KCl (30 mmol/L) was first obtained to check the contractile response of the vascular smooth muscle. The contractile response to KCl constantly ranged between 8 and 10 g with all the arterial segments used in the present study. After washout and equilibration, the aortic rings were precontracted by 0.3 µmol/L NE, a concentration giving {approx}75% of the maximal contraction. The contractile response to NE constantly ranged between 6 and 9 g with all the arterial segments used in the present study, and as determined by ANOVA, no significant differences in the initial contractions to NE were observed after the various preincubation protocols. After precontraction with NE, the rings were relaxed by cumulative additions of either ACh or SNP in the 1-nmol/L to 0.01-mmol/L concentration range. After washout and after a 30-minute recovery period, aortic rings were incubated with either native LDLs, oxidized LDLs, purified LPC, cholesterol, or cholesterol oxides for incubation periods ranging from 15 minutes to 5 hours. The final concentration of LDL protein in the organ bath was constantly 1 mg/mL. The final concentration of pure compounds, including LPC, cholesterol, and cholesterol oxides, ranged from 10 to 120 µg/mL. Control segments were incubated in parallel in Krebs' buffer. At the end of the incubation period, preparations incubated with either native or oxidized LDLs were washed with Krebs' buffer to remove LDLs, and aortic segments were again precontracted with NE and progressively relaxed with either ACh or SNP as described above.

Preparation of Lipid Solutions
Cholesterol and cholesterol oxides (7-ketocholesterol, 7ß-hydroxycholesterol, 5{alpha},6{alpha}-epoxycholesterol, and 19-hydroxycholesterol) were first dissolved in ethanol (20 µL) and then mixed at a 1:1 molar ratio with a solution of fatty acid–poor BSA dissolved in Krebs' buffer.

Light and Electron Microscopy
Aortic segments were fixed for 75 minutes with 4% paraformaldehyde and 1.5% glutaraldehyde prepared in a 0.1-mol/L cacodylate buffer (pH 7.4), postfixed in osmium tetroxide, dehydrated with graded ethanol series, and finally embedded in epoxy resin. Subsequently, either 2-µm semithin or 60- to 90-nm ultrathin sections were obtained from various segments along the descending aorta. The semithin sections were observed by light microscopy after staining with toluidine blue, and endothelium integrity was evaluated by the number and lining of endothelial cells. The ultrathin sections were stained with uranyl acetate and lead citrate and were examined with an electron microscope (model H 600, Hitachi).27

Colorimetric MTT Assay
Cellular metabolism was evaluated by MTT assay28 on bovine aortic endothelial cells cultured in 96-well microculture plates and treated with 7-ketocholesterol as previously described.27 Cellular metabolism was shown to be proportional to absorbance values measured at 540 nm.28

Other Analysis
All chemical assays were performed on a COBAS-BIO centrifugal analyzer (Roche). Total cholesterol, HDL cholesterol, unesterified cholesterol, phospholipids, and triglycerides were measured by enzymatic methods with the use of Boehringer reagents. Apo B concentrations were determined by immunoturbidimetry29 with anti–apo B polyclonal antibodies purchased from Boehringer. Proteins were assayed with the use of bicinchoninic acid reagent (Pierce) according to the method of Smith et al.30

Chemical Compounds
ACh, 5{alpha},6{alpha}-epoxycholestan-3ß-ol (5{alpha},6{alpha}-epoxycholesterol), 5-cholestene-3ß,7ß-diol (7ß-hydroxycholesterol), 5-cholesten-3ß-ol-7-one (7-ketocholesterol), 5-cholestene-3ß,19-diol (19-hydroxycholesterol), cholesterol, epicoprostanol, LPC, phosphatidyl-N,N-dimethylethanolamine-dipalmitoyl, (±)-arterenol hydrochloride (NE), SNP, fatty acid–poor BSA, BHT, and N{omega}-nitro-L-arginine were purchased from Sigma Chemical Co. As indicated by the supplier, cholesterol oxides were a minimum of 98% pure. We confirmed the high degree of purity by analyzing cholesterol oxides by the use of capillary gas chromatography.

Data Analysis
The maximal relaxation (Emax) induced either by ACh or by SNP and expressed as a percentage of the contraction to NE (0.3 µmol/L) was determined from experimental data. pD2 values, corresponding to the negative logarithm of the concentration required to produce a half-maximal relaxing effect (EC50), were calculated after fitting each curve according to a sigmoidal equation of the form

in which X is the agonist concentration; P1, the lower plateau response; P2, the range between the lower and the maximal plateaus of the concentration-effect curve; P3, a negative curvature index indicating the slope independently of the range; and P4, log EC50.31 Emax and pD2 values obtained with arterial rings pretreated with either LDL, cholesterol derivatives, or LPC were compared with corresponding Emax and pD2 values obtained with control rings that were incubated in parallel only in Krebs' buffer.

Data are expressed as mean±SE. Statistical comparison of data means was performed by use of either ANOVA, Student's t test, or Wilcoxon signed rank test as indicated. The coefficient of correlation was calculated by Spearman's rank correlation analysis.


*    Results
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 up arrowIntroduction
 up arrowMethods
 *Results
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Effects of Oxidation on the Composition of LDL Particles
Although {alpha}-tocopherol, {gamma}-tocopherol, {alpha}-carotene, and ß-carotene were present in native LDLs (4.28±0.2, 0.65±0.04, 0.19±0.04, and 1.94±0.22 mg/g of LDL protein, respectively), none of them were detected after the copper-induced oxidation step. The disappearance of antioxidants was accompanied by marked alterations in LDL composition. As shown in Table 1Down, significant decreases in the triglyceride, cholesteryl ester, unesterified cholesterol, and phosphatidylcholine contents of LDL were induced by the oxidation process (Table 1Down). In addition, significant alterations in the overall fatty acid composition of oxidized LDL were observed. The oxidation step did not modify the level of the two major saturated fatty acids, ie, palmitic (16:0) and stearic acids (18:0). In contrast, monounsaturated fatty acids (palmitoleic [16:1] and oleic acids [18:1]) and, to a greater extent, polyunsaturated fatty acids (linoleic [18:2] and arachidonic acids [20:4]) were significantly reduced by oxidation (Table 1Down).


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  		Table 1. Lipid Content and Fatty Acid Composition of LDL Particles

The oxidation-induced decrease among various lipid species of LDLs was associated with increments in several lipid products, including cholesterol oxides, lipoperoxides, and LPC. In accordance with previous studies,32 33 34 35 36 7{alpha}-hydroxycholesterol, 7ß-hydroxycholesterol, and 7-ketocholesterol were the major cholesterol derivatives generated in LDLs during oxidation. Only trace amounts of individual cholesterol derivatives oxidized in position 7 were detected in native LDLs (Table 2Down). In fact, the cholesterol oxide content was increased by {approx}50-fold in oxidized LDLs compared with native counterparts. Oxidation of LDLs was also accompanied by an {approx}44-fold increase in their lipoperoxide content and an {approx}6-fold increase in their LPC content (Table 2Down).


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  		Table 2. Measurement of Oxidation-Derived Compounds in LDL Particles

Effect of Oxidized LDL on Vascular Reactivity
Combined data from distinct experiments with native and oxidized LDLs prepared from 12 human plasma samples are shown in Fig 1Down. No significant difference in the relaxation to ACh was observed between rabbit aorta either incubated without addition or preincubated in the presence of native LDLs. The difference between the maximal relaxation values measured either with control segments or with segments preincubated with native LDLs (94.1±0.8% versus 91.4±1.1%, respectively) as well as the difference between the pD2 values under the same conditions (7.2±0.1 versus 7.1±0.1, respectively) were not statistically significant. In contrast to observations made with native LDLs, the preincubation of rabbit aorta in the presence of oxidized LDLs inhibited the relaxation induced by ACh. Indeed, maximal relaxation to ACh decreased from 94.1±0.8% in control segments to 72.0±6.7% in segments treated with oxidized LDLs (P<.01). In addition, pD2 values decreased from 7.2±0.1 in control segments to 6.6±0.1 in segments treated with oxidized LDLs (P<.001).



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  		Figure 1. Effect of native and oxidized LDLs on the endothelium-dependent relaxation of rabbit aorta induced by ACh. Graph shows cumulative concentration-response curves to ACh in aortas precontracted with 0.3 µmol/L NE and preincubated for 2 hours with either native LDLs (nat-LDL, 1 g protein/L), oxidized LDLs (ox-LDL, 1 g protein/L), or Krebs' buffer (control). Each point represents the mean±SE of 12 distinct experiments.

In accordance with previous data,13 the resting tension of some of the isolated arterial segments studied tended to slightly increase during preincubation in the presence of oxidized LDLs. However, the resting tension of arterial segments returned to the initial value of {approx}2 g after washout (1.7±0.6 g before pretreatment with oxidized LDLs versus 1.8±0.7 g after pretreatment; P=NS).

Among all the subjects studied, the extent of inhibition of endothelium-dependent relaxation varied markedly from one preparation of oxidized LDLs to another, with an observed decrease in maximal relaxation ranging from 0% to 66%. Similar interindividual variations in pD2 values were noted, with an observed decrease in pD2 ranging from 0.21 to 1.3 logarithmic units.

Correlation of Various Components of Oxidized LDL With the Inhibition of the Endothelium-Dependent Relaxation of Rabbit Aorta
The absolute difference between Emax obtained in segments preincubated with oxidized LDLs and Emax obtained in segments preincubated with native LDLs ({Delta}Emax) was compared with the amount of cholesterol oxides, lipoperoxides, and LPC formed in oxidized LDLs. Similarly, we searched for correlations between the difference between pD2 values obtained with native and oxidized LDLs ({Delta}pD2) and the cholesterol oxide content, lipoperoxide content, and LPC content of oxidized LDLs. {Delta}Emax observed between arteries incubated with oxidized and native LDLs correlated significantly with the amount of 7-ketocholesterol (r=.608; P=.044), 7{alpha}-hydroxycholesterol (r=.650; P=.031), and 7ß-hydroxycholesterol (r=.608; P=.044). In contrast, {Delta}Emax did not correlate with the amount of lipoperoxides or LPC. No significant relationships were found between any of the oxidized LDL compounds measured and {Delta}pD2 obtained for arteries incubated with oxidized and native LDLs. These observations suggest, therefore, that cholesterol derivatives oxidized in position 7 but not lipoperoxides or LPC may account for the ability of oxidized LDLs to reduce significantly the maximal relaxation of rabbit aorta to ACh. In contrast, the significant decrease in pD2 values observed with oxidized LDLs did not relate directly to one specific compound generated in oxidized LDLs, suggesting in fact that the decrease in sensitivity of the arteries to ACh does not rely on one single compound identified in oxidized LDLs.

Comparative Effect of Various Cholesterol Oxides on the ACh-Mediated Relaxation of Rabbit Aorta
As shown in Fig 2Down, the concentration-response curve to ACh in NE-precontracted rabbit aorta was modified only by cholesterol derivatives oxidized in position 7 but not by cholesterol or by the other derivatives studied, ie, 19-hydroxycholesterol and 5{alpha},6{alpha}-epoxycholesterol. Over the 0- to 120-µg/mL concentration range studied, 7-ketocholesterol and 7ß-hydroxycholesterol reduced gradually and significantly the Emax of rabbit aorta (Table 3Down). However, in agreement with the absence of correlations between the cholesterol oxide content of oxidized LDLs and their ability to modify the sensitivity of aorta to ACh, pD2 values were affected neither by 7ß-hydroxycholesterol nor by 7-ketocholesterol (Table 3Down).



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  		Figure 2. Effect of cholesterol and various cholesterol oxides on the endothelium-dependent relaxation of rabbit aorta induced by ACh. Graph shows cumulative concentration-response curves to ACh in aortas precontracted with 0.3 µmol/L NE and preincubated for 2 hours with 60 µg/mL of either 7-ketocholesterol (7-keto), 7ß-hydroxycholesterol (7ß-OH), 5{alpha},6{alpha}-epoxycholesterol (5{alpha},6{alpha}-epoxy), 19-hydroxycholesterol (19-OH), or cholesterol, or in the presence of Krebs' buffer (control). Each point represents the mean±SE of 9 to 33 distinct experiments.


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  		Table 3. Comparative Effects of Cholesterol and Cholesterol Oxides on Maximal Relaxation (Emax) and Sensitivity (pD2) of Rabbit Aortic Rings to ACh

Fig 3Down shows the time-dependent effect of 7-ketocholesterol (60 µg/mL) on the ACh-mediated relaxation of isolated rabbit aortas. The cumulative addition of ACh progressively relaxed the NE-precontracted segments, which were maintained for 5 hours at 37°C with no cholesterol oxide added. The ACh-mediated, endothelium-dependent relaxation of aortic segments was significantly reduced in a time-dependent manner in the presence of 7-ketocholesterol as early as 1 hour after beginning the incubation period. After a 5-hour preincubation at 37°C in the presence of 7-ketocholesterol, a contraction in place of a dilation of aortic rings was observed in response to cumulative concentrations of ACh (Fig 3Down).



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  		Figure 3. Time-dependent effect of 7-ketocholesterol (7-keto) on the endothelium-dependent relaxation of rabbit aorta induced by ACh. Graph shows cumulative concentration-response curves to ACh in one aorta precontracted with 0.3 µmol/L NE and preincubated for either 1, 2, 3, or 5 hours with 60 µg/mL 7-ketocholesterol. Control segments were incubated for 5 hours in the presence of Krebs' buffer.

Histological Evaluation of Cholesterol Oxide–Treated Rabbit Aorta
Light microscopy did not reveal alterations in the number and lining of endothelial cells and smooth muscle cells in arterial segments incubated for 2 hours with 7-ketocholesterol (final concentration, 90 µg/mL). Indeed, after staining of semithin arterial slices with toluidine blue, the mean number of endothelial cells obtained from nine control, untreated arterial sections and nine 7-ketocholesterol–treated arterial sections was identical (213±26 cells/section and 217±18 cells/section, respectively). Consistent observations were made with four control arteries and four treated arteries (results not shown). With the use of transmission electron microscopy, no ultrastructural alterations in endothelial cells or smooth muscle cells were observed in either control or 7-ketocholesterol–treated arteries (Fig 4Down). In both cases, endothelial cells formed a continuous sheet lining the lumen, and the cytoplasm was structurally intact, with a few vacuoles and with normal mitochondria (Fig 4Down). Finally, as shown in Fig 4Down, tight junctions were structurally intact.



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  		Figure 4. Transmission electron micrographs of rabbit aortas treated or not treated with 7-ketocholesterol. Segments of rabbit aorta were incubated for 2 hours at 37°C in the absence (A and B) or presence (C and D) of 7-ketocholesterol (final concentration, 90 µg/mL). EC indicates endothelial cell; IEL, internal elastic lamina; and SMC, smooth muscle cell. Scale bars=1 µm.

Evaluation of Cellular Metabolism in Cultured Bovine Aortic Endothelial Cells
Mean absorbances measured with MTT assay in bovine aortic endothelial cells treated or not for 4 hours with 80 µg/mL 7-ketocholesterol did not differ significantly (0.412±0.007 and 0.393±0.010, respectively; n=3), indicating no variations in cellular metabolism.

Effect of LPC on the ACh-Mediated Relaxation of Rabbit Aorta
When the effect of LPC (concentration range, 60 to 120 µg/mL) on the ACh-mediated relaxation of rabbit aorta was investigated in parallel to cholesterol oxides, a significant, concentration-dependent inhibition of the maximal relaxation was also observed. Emax values decreased from 95.5±1.1% in control segments to 44.4±14.5% in segments pretreated with 60 µg/mL LPC and to 20.0±6.6% in segments pretreated with 120 µg/mL LPC (n=4). In addition, LPC decreased the sensitivity of rabbit aorta to ACh. Indeed, the pD2 decreased from 7.27±0.15 in control segments to values <6 for 60 µg/mL and <5.5 for 120 µg/mL.

Combined Effect of 7-Ketocholesterol and LPC on the ACh-Mediated Relaxation of Rabbit Aorta
As shown in Fig 5Down, and in accordance with other data presented herein, 7-ketocholesterol reduced the maximal relaxation of rabbit aortic rings (93.0±1.8% in control arteries versus 75.2±6.6% in arteries treated with 7-ketocholesterol), with no change in pD2 values (7.0±0.3 in control arteries versus 7.0±0.4 in arteries treated with 7-ketocholesterol). The 15-minute treatment of arterial segments with LPC reduced both Emax (93.0±1.8% in control arteries versus 64.1±18.4% in LPC arteries) and pD2 (7.0±0.3 in control arteries versus 5.9±0.3 in LPC arteries). In experiments cumulating the effects of 7-ketocholesterol and LPC, an even greater reduction in Emax values was observed (27.1±21.4%) (Fig 5Down). Due to the nonsigmoidal shape of the curve, the pD2 value corresponding to 7-ketocholesterol plus LPC could not be determined.



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  		Figure 5. Combined effect of 7-ketocholesterol and LPC on the endothelium-dependent relaxation of rabbit aorta induced by ACh. Graph shows cumulative concentration-response curves to ACh in three NE-precontracted aortas that were preincubated either for 2 hours in Krebs' buffer (Control), for 2 hours with 90 µg/mL 7-ketocholesterol (7-keto), for 15 minutes with 10 µg/mL LPC, or for 2 hours with 90 µg/mL of 7-ketocholesterol followed by a 15-minute incubation with 10 µg/mL LPC (7-keto+LPC). Each point represents the mean of three experiments.

Effect of 7-Ketocholesterol on the Endothelium-Independent Relaxation to SNP
As shown in Fig 6Down, preincubation of aortic segments for 2 hours at 37°C in the presence of 7-ketocholesterol (60 µg/mL) did not affect SNP-mediated, endothelium-independent relaxation. In control and 7-ketocholesterol–treated aortic segments, Emax values (95.1±2.0% versus 94.4±1.7%, respectively) and pD2 values (7.4±0.2 versus 7.4±0.1, respectively) did not vary significantly.



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  		Figure 6. Effect of 7-ketocholesterol on the endothelium-independent relaxation evoked by SNP. Graph shows cumulative concentration-response curves to SNP in aortas precontracted with 0.3 µmol/L NE and preincubated for 2 hours with 7-ketocholesterol (7-keto, 60 µg/mL) or in the presence of Krebs' buffer (control). Each point represents the mean±SE of six distinct experiments.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The endothelium of blood vessels plays a central role in the regulation of vascular tone by the release of endothelium-derived relaxing and contracting factors.19 37 38 39 Hypercholesterolemia has been shown to be associated with a marked reduction of the relaxing response to ACh in rabbit,7 40 41 42 monkey,43 and human arteries8 44 45 . The attenuation of normal endothelium-dependent relaxation, associated with hypercholesterolemia and more specifically with high oxidized-LDL levels,14 16 has been demonstrated to occur at an early stage of the disease, before the formation of atherosclerotic lesions.8 9 10 Taken together, previous studies therefore led to the hypothesis that specific compounds of oxidized LDL may act prematurely on endothelial cells by modifying their relaxing function.

Oxidation is known to induce significant changes in the structure and composition of LDL particles, including the loss of antioxidants, the peroxidation of unsaturated fatty acids, the generation of cholesterol oxides, and the alteration in apo B immunoreactivity.46 In the present study, LDLs isolated from normolipidemic human plasma samples were oxidized in vitro with copper sulfate, a widely used oxidation method that induced marked alterations in the antioxidant and lipid content of LDL, as expected.46 Subsequently, the modulating potency of human oxidized LDL on vascular reactivity was quantified with a biological system based on the measurement of the relaxing response of isolated rabbit aortic segments to ACh, an endothelium-dependent relaxing compound. In accordance with previous studies,13 14 15 17 oxidized LDLs were able to inhibit markedly the endothelium-dependent arterial relaxation. Interestingly, disparities appeared in the ability of individual oxidized LDL preparations to act as inhibitors of the endothelium-dependent relaxation of rabbit aorta. Indeed, whereas some oxidized LDL preparations did not promote significant alterations in vascular reactivity, others were able to reduce markedly both the maximal endothelium-dependent relaxation and the sensitivity to ACh of arterial segments. A similar fluctuation in the inhibiting potency of oxidized LDLs from various subjects has been reported by Jacobs and coworkers.11 In the present study, we further attempted to connect variations in the inhibitory potency of oxidized LDLs with modifications in their lipid content. To this end, the main compounds generated during LDL oxidation, including LPC, lipoperoxides, and cholesterol oxides, were assayed and were compared with the ability of LDL particles to inhibit the endothelium-dependent relaxation of rabbit aorta.

In previous studies,14 16 18 the inhibition of relaxation by oxidized LDLs has been attributed to LPC. This conclusion was reached because 1-palmitoyl LPC as well as phospholipase A2–treated LDLs were shown to inhibit endothelium-dependent relaxation.14 16 18 In addition, in support of a role of LPC in mediating the inhibitory effect of oxidized LDL, the reduction of the LPC content of oxidized LDL with delipidated albumin was reported to attenuate the inhibitory effect of LDL on endothelium-dependent relaxation.14 18 However, LPC may not represent the most important factor responsible for the inhibition of artery relaxation by oxidized LDL. In the present study, although a potent, concentration-dependent inhibition of arterial relaxation by pure LPC was observed, we did not find a significant correlation between the amount of LPC formed in oxidized LDLs and their ability to reduce the endothelium-dependent relaxation. These latter observations confirmed data of Plane and coworkers,17 who reported that the LPC content of oxidized LDL was unrelated to the inhibition of the relaxation, suggesting that inhibitory substances other than LPC are present in oxidized LDL. Similarly, studies by Tanner and coworkers,15 in which LPC was not able to mimic the oxidized LDL–mediated inhibition of the endothelium-dependent relaxation in porcine coronary arteries, do not favor a preponderant role of LPC in reducing arterial relaxation. In fact, whereas Kugiyama et al14 and Mangin et al18 demonstrated that the inhibitor(s) of arterial relaxation is (are) localized in the lipid extract of oxidized LDL, the precise role of other lipid components of oxidized LDL, among them cholesterol oxides, was not addressed. Cholesterol oxides comprise a wide family of molecules that result not only from oxidation of LDL, but also from auto-oxidation of cholesterol in air47 48 or from the enzymatic transformation of cholesterol in various cell species.49 In addition, cholesterol oxides have been shown to be present in a number of foodstuffs, including dried egg products, powdered milk, and cheese, as well as in a variety of high-temperature dried animal products.48 50 There is growing evidence for a specific role of cholesterol oxides in the initiation and/or progression of atherosclerosis51 : toxic effects of cholesterol oxides have been reported in the three major cell types of the artery wall, ie, endothelial cells,52 53 smooth muscle cells,54 and fibroblasts,55 and cholesterol oxide levels have been found to be abnormally elevated in hypercholesterolemic plasma and atherosclerotic aortas from rabbits and humans.56 57 58 In accordance with previous studies,34 35 36 we found that copper oxidation of human LDL preparations leads to the formation of three main cholesterol oxides, ie, 7{alpha}-hydroxycholesterol, 7ß-hydroxycholesterol, and 7-ketocholesterol. We observed that only cholesterol oxides and not lipoperoxides or LPC correlate significantly with the reduction in the maximal relaxation induced by ACh in rabbit aorta that were preincubated with oxidized LDLs. In contrast, the cholesterol oxide content of oxidized LDLs did not correlate with their ability to decrease the sensitivity of the arteries to ACh. To confirm further that cholesterol oxides constitute new candidates in mediating the inhibitory effect of oxidized LDL, we investigated whether individual cholesterol oxides could affect the endothelium-dependent relaxation of rabbit aorta. It results that only derivatives of cholesterol oxidized in position 7, ie, 7-ketocholesterol and 7ß-hydroxycholesterol, but not cholesterol or the other cholesterol oxides studied are potent inhibitors of the endothelium-dependent relaxation of rabbit aorta. In agreement with the correlation between the cholesterol oxide content of oxidized LDLs and their ability to reduce the maximal relaxation, 7ß-hydroxycholesterol and 7-ketocholesterol reduced the Emax of rabbit arterial segments but not their sensitivity to ACh (pD2), which suggests that additional factors may account for the significant decrease in pD2 values observed with oxidized LDLs. Interestingly, the vascular effects of 7ß-hydroxycholesterol and 7-ketocholesterol present strong similarities with previous data obtained either in vivo in coronary arteries from hypercholesterolemic patients8 and in vitro in aortic and pulmonary arteries isolated from hypercholesterolemic rabbits.40 In particular, it appears that the endothelium-dependent relaxation to ACh can be either diminished, abolished, or even changed to a contraction both as a result of hypercholesterolemia in vivo8 43 or in response to cholesterol oxides in vitro (present study). Moreover, neither hypercholesterolemia6 9 43 nor preincubation with cholesterol oxides (present study) could affect the arterial relaxation mediated by SNP, an endothelium-independent vasodilator. This latter observation indicates that cholesterol oxides, like hypercholesterolemia, inhibit arterial relaxation by an endothelium-dependent mechanism rather than through a direct effect on underlying smooth muscle cells. The ability of cholesterol derivatives oxidized in position 7 to inhibit the endothelium-dependent relaxation of rabbit aorta after 2 hours of incubation was not mediated through the known cytotoxic effect they may exert on endothelial cells during much longer incubation periods, as recently demonstrated in our laboratory.27 Indeed, in the present study, no evidence for alteration in the structure of endothelial cells and smooth muscle cells could be observed by use of both light and electron microscopy. Finally, the cholesterol oxide–mediated inhibition of prostaglandin I2 synthesis by endothelial cells59 is unlikely to account in the present study for the reduced ability of endothelial cells to relax smooth muscle cells because the ACh-mediated relaxation of rabbit aorta (1) was totally inhibited with N{omega}-nitro-L-arginine, an inhibitor of NO synthase, and (2) was not affected by indomethacin, an inhibitor of cyclooxygenase (results not shown).

In conclusion, the results of the present study demonstrated that cholesterol derivatives oxidized at position 7 constitute potent inhibitors of endothelium-dependent arterial relaxation. Because cholesterol oxides have been identified in a number of foodstuffs and human tissues, and because their ability to inhibit arterial relaxation was observed at levels compatible with those reported in hypercholesterolemic plasma56 57 and in atherosclerotic lesions,57 58 they may contribute significantly to the occurrence of vasospasm at an early stage of atherosclerotic disease.


*    Selected Abbreviations and Acronyms
 
ACh = acetylcholine
apo = apolipoprotein
EC50 = half-maximal relaxing effect
Emax = maximal relaxation
HPLC = high-performance liquid chromatography
LPC = {alpha}-lysophosphatidylcholine
MTT = methylthiazol tetrazolium
NE = norepinephrine
NO = nitric oxide
pD2 = negative logarithm of the concentration required to produce a half-maximal relaxing effect
SNP = sodium nitroprusside
TBS = Tris buffer


*    Acknowledgments
 
This work was supported by the Universite de Bourgogne, the Conseil Regional de Bourgogne, the Institut National de la Sante et de la Recherche Medicale (INSERM), the Fondation de France, and the Comite Francais de Coordination des Recherches sur l'Atherosclerose et le Cholesterol (ARCOL). The technical assistance of Maryvonne Moisant in electron microscopy analysis is greatly appreciated.

Received May 7, 1996; revision received September 11, 1996; accepted September 24, 1996.


*    References
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  1. Henry PD, Yokoyama M. Supersensitivity of atherosclerotic rabbit aorta to ergonovine. J Clin Invest. 1980;66:306-313.
  2. Yokoyama M, Akita H, Mizutani T, Fukuzaki H, Watanabe Y. Hyperreactivity of coronary arterial smooth muscles in response to ergonovine from rabbits with hereditary hyperlipidemia. Circ Res. 1983;53:63-71.[Abstract/Free Full Text]
  3. Heistad DD, Armstrong ML, Marcus ML, Piegors DJ, Mark AL. Augmented responses to vasoconstrictor stimuli in hypercholesterolemic and atherosclerotic monkeys. Circ Res. 1984;54:711-718.[Abstract/Free Full Text]
  4. Merkel LA, Rivera LM, Bilder GE, Perrone MH. Differential alteration of vascular reactivity in rabbit aorta with modest elevation of serum cholesterol. Circ Res. 1990;67:550-555.[Abstract/Free Full Text]
  5. Galle J, Busse R, Bassenge E. Hypercholesterolemia and atherosclerosis change vascular reactivity in rabbits by different mechanisms. Arterioscler Thromb. 1991;11:1712-1718.[Abstract/Free Full Text]
  6. Verbeuren TJ, Jordaens FH, Zonnekeyn LL, Van Hove CE, Coene M-C, Herman AG. Effect of hypercholesterolemia on vascular reactivity in the rabbit, I: endothelium-dependent and endothelium-independent contractions and relaxations in isolated arteries of control and hypercholesterolemic rabbits. Circ Res. 1986;58:552-564.[Abstract/Free Full Text]
  7. Jayakody L, Senaratne M, Thomson A, Kappagoda T. Endothelium-dependent relaxation in experimental atherosclerosis in the rabbit. Circ Res. 1987;60:251-264.[Abstract/Free Full Text]
  8. Zeiher AM, Drexler H, Wollschlager H, Just H. Modulation of coronary vasomotor tone in humans: progressive endothelial dysfunction with different early stages of coronary atherosclerosis. Circulation. 1991;83:391-401.[Abstract/Free Full Text]
  9. Reddy KG, Nair RN, Sheehan HM, Hodgson JM. Evidence that selective endothelial dysfunction may occur in the absence of angiographic or ultrasound atherosclerosis in patients with risk factors for atherosclerosis. J Am Coll Cardiol. 1994;23:833-843.[Abstract]
  10. Celermajer DS, Sorensen KE, Bull C, Robinson J, Deanfield JE. Endothelium-dependent dilation in the systemic arteries of asymptomatic subjects relates to coronary risk factors and their interaction. J Am Coll Cardiol. 1994;24:1468-1474.[Abstract]
  11. Jacobs M, Plane F, Bruckdorfer RK. Native and oxidized low-density lipoproteins have different inhibitory effects on endothelium-derived relaxing factor in the rabbit aorta. Br J Pharmacol. 1990;100:21-26.[Medline] [Order article via Infotrieve]
  12. Galle J, Mulsch A, Busse R, Bassenge E. Effects of native and oxidized low density lipoproteins on formation and inactivation of endothelium-derived relaxing factor. Arterioscler Thromb. 1991;11:198-203.[Abstract/Free Full Text]
  13. Simon BC, Cunningham LD, Cohen RA. Oxidized low density lipoproteins cause contraction and inhibit endothelium-dependent relaxation in the pig coronary artery. J Clin Invest. 1990;86:75-79.
  14. Kugiyama K, Kerns SA, Morrisett JD, Roberts R, Henry PD. Impairment of endothelium-dependent arterial relaxation by lysolecithin in modified low-density lipoproteins. Nature. 1990;344:160-162.[Medline] [Order article via Infotrieve]
  15. Tanner FC, Noll G, Boulanger CM, Luscher TF. Oxidized low-density lipoproteins inhibit relaxations of porcine coronary arteries: role of scavenger receptor and endothelium-derived nitric oxide. Circulation. 1991;83:2012-2020.[Abstract/Free Full Text]
  16. Yokoyama M, Hirata K-I, Miyake R, Akita H, Ishikawa Y, Fukuzaki H. Lysophosphatidylcholine: essential role in the inhibition of endothelium-dependent vasorelaxation by oxidized low density lipoprotein. Biochem Biophys Res Commun. 1990;168:301-308.[Medline] [Order article via Infotrieve]
  17. Plane F, Bruckdorfer RK, Kerr P, Steuer A, Jacobs M. Oxidative modification of low-density lipoproteins and the inhibition of relaxations mediated by endothelium-derived nitric oxide in rabbit aorta. Br J Pharmacol. 1992;105:216-222.[Medline] [Order article via Infotrieve]
  18. Mangin EL, Kugiyama K, Nguy JH, Kerns SA, Henry PD. Effects of lysolipids and oxidatively modified low-density lipoprotein on endothelium-dependent relaxation of rabbit aorta. Circ Res. 1993;72:161-166.[Abstract/Free Full Text]
  19. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980;288:373-376.[Medline] [Order article via Infotrieve]
  20. Moncada S, Radomski MW, Palmer RMJ. Endothelium-derived relaxing factor. Biochem Pharmacol. 1988;37:2495-2501.[Medline] [Order article via Infotrieve]
  21. El-Saadani M, Esterbauer H, El-Sayed M, Goher M, Nassar AY, Jurgens G. A spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent. J Lipid Res. 1989;30:627-630.[Abstract]
  22. Park SW, Addis PB. Capillary column gas-liquid chromatographic resolution of oxidized cholesterol derivatives. Anal Biochem. 1985;149:275-283.[Medline] [Order article via Infotrieve]
  23. Hax WMA. High-performance liquid chromatographic separation and photometric detection of phospholipids. J Chromatogr. 1977;142:735-741.[Medline] [Order article via Infotrieve]
  24. Shantha NC. Gas chromatography of fatty acids. J Chromatogr. 1992;624:37-51.[Medline] [Order article via Infotrieve]
  25. Miller KW, Yang CS. An isocratic high-performance liquid chromatography method for the simultaneous analysis of plasma retinol, {alpha}-tocopherol, and various carotenoids. Anal Biochem. 1985;145:21-26.[Medline] [Order article via Infotrieve]
  26. Andrews HE, Bruckdorfer KR, Dunn RC, Jacobs M. Low density lipoproteins inhibit endothelium-dependent relaxation in rabbit aorta. Nature. 1980;327:237-239.
  27. Lizard G, Deckert V, Dubrez L, Moisant M, Gambert P, Lagrost L. Induction of apoptosis in endothelial cells treated with cholesterol oxides. Am J Pathol. 1996;148:16125-16138.
  28. Mossman T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. J Immunol Methods. 1983;65:55-63.[Medline] [Order article via Infotrieve]
  29. Rifai N, King ME. Immunoturbidimetric assays of apolipoproteins A, AI, AII and B in serum. Clin Chem. 1986;32:957-961.[Abstract/Free Full Text]
  30. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985;150:76-85.[Medline] [Order article via Infotrieve]
  31. Deckert V, Pruneau D, Elghozi J-L. Mediation by 5-HT1D receptors of 5-hydroxytryptamine-induced contractions of rabbit middle and posterior cerebral arteries. Br J Pharmacol. 1994;112:939-945.[Medline] [Order article via Infotrieve]
  32. Zhang H, Basra HJK, Steinbrecher UP. Effects of oxidatively modified LDL on cholesterol esterification in cultured macrophages. J Lipid Res. 1990;31:1361-1369.[Abstract]
  33. Jialal I, Freeman DA, Grundy SM. Varying susceptibility of different low-density lipoproteins to oxidative modification. Arterioscler Thromb. 1991;11:482-488.[Abstract/Free Full Text]
  34. Bhadra S, Arshad MAQ, Rymaszewski Z, Norman E, Wherley R, Subbiah MTR. Oxidation of cholesterol moiety of low density lipoprotein in the presence of human endothelial cells or Cu2+ ions: identification of major products and their effects. Biochem Biophys Res Commun. 1991;176:431-440.[Medline] [Order article via Infotrieve]
  35. Tamasawa N, Takebe K. Analytical examination of oxidized free and esterified 7-ketocholesterol and related oxysterols in human plasma incubated with copper. Tohoku J Exp Med. 1992;168:37-45.[Medline] [Order article via Infotrieve]
  36. Malavasi B, Rasetti MF, Roma P, Fogliatto R, Allevi P, Catapano AL, Galli G. Evidence for the presence of 7-hydroperoxycholest-5-en-3ß-ol in oxidized human LDL. Chem Phys Lipids. 1992;62:209-214.[Medline] [Order article via Infotrieve]
  37. Vanhoutte PM, Luscher TF, Graser T. Endothelium-dependent contractions. Blood Vessels. 1991;28:74-83.[Medline] [Order article via Infotrieve]
  38. Shepherd JT, Katusic ZS. Endothelium-derived vasoactive factors, I: endothelium-dependent relaxation. Hypertension. 1991;18(suppl III):III-76-III-85.
  39. Luscher TF, Boulanger CM, Yang Z, Noll G, Dohi Y. Interactions between endothelium-derived relaxing and contracting factors in health and cardiovascular disease. Circulation. 1993;87(suppl V):V-36-V-44.
  40. Ibengwe JK, Suzuki H. Changes in mechanical responses of vascular smooth muscles to acetylcholine, noradrenaline and high-potassium solution in hypercholesterolemic rabbits. Br J Pharmacol. 1986;87:395-402.[Medline] [Order article via Infotrieve]
  41. Osborne JA, Siegman MJ, Sedar AW, Mooers SU, Lefer AM. Lack of endothelium-dependent relaxation in coronary resistance arteries of cholesterol-fed rabbits. Am J Physiol. 1989;256:C591-C597.[Abstract/Free Full Text]
  42. Verbeuren TJ, Jordaens FH, Van Hove CE, Van Hoydonck A-E, Herman AG. Release and vascular activity of endothelium-derived relaxing factor in atherosclerotic rabbit aorta. Eur J Pharmacol. 1990;191:173-184.[Medline] [Order article via Infotrieve]
  43. Sellke FW, Armstrong ML, Harrison DG. Endothelium-dependent vascular relaxation is abnormal in the coronary microcirculation of atherosclerotic primates. Circulation. 1990;81:1586-1593.[Abstract/Free Full Text]
  44. Creager MA, Cooke JP, Mendelsohn ME, Gallagher SJ, Coleman SM, Loscalzo J, Dzau VJ. Impaired vasodilation of forearm resistance vessels in hypercholesterolemic humans. J Clin Invest. 1990;86:228-234.
  45. Bossaler C, Habib GB, Yamamoto H, Williams C, Wells S, Henry PD. Impaired muscarinic endothelium-dependent relaxation and cyclic guanosine 5'-monophosphate formation in atherosclerotic human coronary artery and rabbit aorta. J Clin Invest. 1987;79:170-174.
  46. Esterbauer H, Dieber-Rotheneder M, Waeg G, Striegl G, Jurgens G. Biochemical, structural, and functional properties of oxidized low-density lipoprotein. Chem Res Toxicol. 1990;3:77-92.[Medline] [Order article via Infotrieve]
  47. Sevanian A, Peterson A, Trosko J. The formation and cytotoxic properties of cholesterol oxides. J Am Oil Soc. 1985;62:635. Abstract.
  48. Smith LL. Cholesterol auto-oxidation. Chem Phys Lipids. 1987;44:87-125.[Medline] [Order article via Infotrieve]
  49. Martin CM, Nicholas HJ. Metabolism of cholesteryl palmitate by rat brain in vitro: formation of cholesterol epoxides and cholestan-3ß,5{alpha},6ß-triol. J Lipid Res. 1973;14:618-624.[Abstract]
  50. Addis PB. Occurrence of lipid oxidation products in foods. Food Chem Toxicol. 1986;24:1021-1030.[Medline] [Order article via Infotrieve]
  51. Imai H, Werthessen NT, Subramanyam V, Lequesne PW, Soloway AH, Kanisawa M. Angiotoxicity of oxygenated sterols and possible precursors. Science. 1980;207:651-653.[Abstract/Free Full Text]
  52. Sevanian A, Berliner J, Peterson H. Uptake, metabolism, and cytotoxicity of isomeric cholesterol-5,6-epoxides in rabbit aortic endothelial cells. J Lipid Res. 1991;32:147-155.[Abstract]
  53. Hennig B, Boissonneault GA. Cholestan-3ß,5{alpha},6ß-triol decreases barrier function of cultured endothelial cell monolayers. Atherosclerosis. 1987;68:255-261.[Medline] [Order article via Infotrieve]
  54. Peng S-K, Tham P, Taylor BC, Mikkelson B. Cytotoxicity of oxidation derivatives of cholesterol on cultured aortic smooth muscle cells and their effect on cholesterol biosynthesis. Am J Clin Nutr. 1979;32:1033-1042.[Abstract/Free Full Text]
  55. Highley NA, Taylor SL. The steatotic and cytotoxic effects of cholesterol oxides in cultured cells. Food Chem Toxicol. 1984;22:983-992.[Medline] [Order article via Infotrieve]
  56. Gray MF, Lawrie TDV. Isolation and identification of cholesterol {alpha}-oxide and other minor sterols in human serum. Lipids. 1971;6:836-843.[Medline] [Order article via Infotrieve]
  57. Hodis HN, Crawford DW, Sevanian A. Cholesterol feeding increases plasma and aortic tissue cholesterol oxide levels in parallel: further evidence for the role of cholesterol oxidation in atherosclerosis. Atherosclerosis. 1991;89:117-126.[Medline] [Order article via Infotrieve]
  58. Carpenter KLH, Taylor SE, Ballantine JA, Fussell B, Halliwell B, Mitchinson MJ. Lipids and oxidised lipids in human atheroma and normal aorta. Biochim Biophys Acta. 1993;1167:121-130.[Medline] [Order article via Infotrieve]
  59. Peng SK, Hu B, Peng AY, Morin RJ. Effect of cholesterol oxides on prostacyclin production and platelet adhesion. Artery. 1993;20:122-134.[Medline] [Order article via Infotrieve]



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