(Stroke. 1997;28:2266-2272.)
© 1997 American Heart Association, Inc.
Articles |
Mildly Oxidized Low-Density Lipoprotein Impairs Responses of Carotid but Not Basilar Artery in Rabbits
From the Cardiovascular Center and Center on Aging, Departments of Internal Medicine and Pharmacology, University of Iowa, Iowa City (R.P., F.M.F., H.T., D.D.H.), and the Department of Clinical and Experimental Medicine, Federico II University of Naples (Italy) (C.N., A.P.).
Correspondence to Donald D. Heistad, MD, Department of Internal Medicine, University of Iowa, 200 Hawkins Dr, Iowa City, IA 52242-1081. E-mail donald-heistad{at}uiowa.edu
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Abstract Background and Purpose Intracranial blood vessels appear to be relatively resistant to development of atherosclerosis. The goal of this study was to compare effects of mildly oxidized LDL (ox-LDL) on endothelium-dependent responses of intracranial and extracranial arteries in vitro.
Methods LDL was purified from plasma of healthy subjects and mildly oxidized by means of a xanthine/xanthine oxidase reaction. Contraction of the rabbit basilar and carotid arteries in response to histamine and phenylephrine and relaxation in precontracted vessels to acetylcholine and sodium nitroprusside were evaluated after 30 minutes of exposure to LDL or ox-LDL (100 µg/mL).
Results Exposure to LDL had little or no effect on vascular responses. After exposure to ox-LDL, contraction to histamine and phenylephrine and relaxation to acetylcholine were impaired significantly in carotid (P<.05) but not in basilar artery. Relaxation to sodium nitroprusside was not significantly impaired by ox-LDL in the basilar artery. In the carotid artery, relaxation to sodium nitroprusside was significantly impaired by ox-LDL when the vessels were precontracted with phenylephrine but not histamine. Impairment of vascular responses by ox-LDL was prevented by addition of superoxide dismutase, catalase, or dimethylthiourea to the LDL solution before addition of xanthine/xanthine oxidase.
Conclusions Mildly ox-LDL impairs contraction and endothelium-dependent relaxation in the carotid but not in basilar artery. Thus, intracranial arteries may be relatively resistant to mildly ox-LDL.
Key Words: basilar artery • carotid arteries • endothelium • lipoproteins, LDL cholesterol • oxygen radical • rabbits
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Introduction |
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Hypercholesterolemia is a risk factor for coronary vascular disease, but the role of hypercholesterolemia as a risk factor for cerebrovascular disease is still debated.1 Studies in primates and rabbits indicate that atherosclerosis is less severe in intracranial than extracranial and coronary arteries during hypercholesterolemia.1,2 Similarly, in humans it appears that intracerebral atherosclerosis occurs much later than coronary atherosclerosis.1,3
Macrophage-derived foam cells, which contain LDL, accumulate in atherosclerotic lesions.4 Modification of LDL, induced by acetylation or oxidation, stimulates uptake of LDL through the macrophage scavenger receptor(s) and appears to play a pivotal role in the pathophysiology of atherosclerosis.5,6 Oxidative modification of LDL may be triggered by generation of reactive oxygen species in vivo.7-9
Endothelial dysfunction is an early manifestation of atherosclerosis.10 High concentrations of n-LDL inhibit endothelium-dependent relaxation11,12 by a rapid and reversible mechanism.13 ox-LDL is more potent than n-LDL in producing impairment of endothelial function in rings of aorta and coronary arteries.12-16
ox-LDL isolated directly from atherosclerotic arteries appears to be similar to lipoproteins oxidized in vitro with copper sulfate.17 Oxidation of LDL by copper sulfate, which involves reactions of preformed hydroperoxides,18 is achieved at micromolar concentrations that appear to be several-fold higher than pathophysiological concentrations.19 We therefore have used the X/XO reaction to generate reactive oxygen species, which appear to be comparable in magnitude to those induced in pathophysiological conditions7-9 and produce mild LDL oxidation similar to that which may occur in vivo.
The goal of the study was to test the effects of ox-LDL generated by X/XO reaction on reactivity of isolated arteries in vitro and to compare the effects of ox-LDL on an intracranial (basilar) versus an extracranial (carotid) artery.
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Materials and Methods |
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LDL Purification and Oxidation
Blood was obtained from healthy, normolipidemic, and nonsmoking men after an overnight fast. No subjects were taking drugs or vitamin supplements at the time of the study. LDL was isolated and purified from plasma by density ultracentrifugation with a vertical rotor VTi 50 (Beckman) in a KBr gradient modified as previously described.20-23 Briefly, freshly collected plasma was adjusted to a density of 1.225 kg/L by addition of 0.345 g KBr. Isotonic saline (d=1.006 kg/L) containing EDTA (1 mmol/L, final concentration) was layered on top of plasma in a polymer centrifuge tube (Beckman Instruments). The tubes were centrifuged at 45 500 rpm for 2.5 hours at 10°C. A second purification was achieved by ultracentrifugation at 68 000 rpm for 40 minutes at 10°C, followed by a Sephacryl S-300 column to desalt and purify the final sample. LDL was used within a few hours after isolation to minimize spontaneous oxidation. Protein content was measured by the assay of Lowry et al 24 with bovine serum albumin used as a standard. The cholesterol content of LDL was measured by enzymatic assay (Cholesterol 50, Sigma).
Reactive oxygen species were generated by means of the enzymatic reaction of xanthine with xanthine oxidase in 150 mmol/L NaCl/0.01 mol/L sodium phosphate buffer without EDTA, pH 7.4, as described previously.20-22 LDL was incubated for 6 hours at 37°C in the presence of xanthine (2 mmol/L, final concentration) and xanthine oxidase (Boehringer-Mannheim, 100 mU/mL, salicylate-free, from bovine milk, specific activity 1 U/mg of protein) in 0.150 mol/L NaCl/0.01 mol/L sodium phosphate at pH 7.4. After incubation for 6 hours, further LDL modifications were prevented by addition of the chain breaker butylated hydroxytoluene (100 µmol/L final concentration) to the tube with the reaction. In experiments in which n-LDL was used as a control, butylated hydroxytoluene was added to the test tube before the incubation.
The X/XO reaction yields both superoxide radicals and hydrogen peroxide,25,26 which in turn may give rise to hydroxyl radicals in the presence of trace amounts of iron or other transition heavy metals. This system generates approximately 20 nmol/min per milliliter of superoxide radicals and approximately 40 nmol/min per milliliter of hydrogen peroxide at peak activity (ie, 90 seconds), which then progressively declines and arrests within 6 minutes.20-22 Therefore, we examined the role of several reactive oxygen species on LDL oxidation by performing parallel experiments in the presence of scavengers of reactive oxygen species. Scavengers were added to the LDL solution immediately before generation of reactive oxygen species. SOD (CuZn SOD from bovine erythrocytes, specific activity 3570 U/mg of protein) was added to a final concentration of 330 U/mL. Catalase (Boehringer Mannheim, from bovine liver, specific activity 40 000 U/mg of protein), which degrades hydrogen peroxide, was added at 1000 U/mL. Final concentrations of the hydroxyl radical scavenger DMTU were 10 mmol/L. We have described detailed characterization of LDL modified by X/XO reaction previously.20-22
Experimental Preparation
New Zealand White rabbits (weight, 2.5 to 3.5 kg) were given a
lethal dose of sodium pentobarbital (100 mg/kg IV), and basilar
and carotid arteries were removed. Connective tissue was removed from
the carotid artery. The excised tissues were placed in Krebs' buffer
(in mmol/L): NaCl 118.3, KCl 4.7, CaCl2 2.5,
MgSO4 1.2, KH2PO4 1.2,
NaHCO3 25, EDTA calcium 0.026, and glucose 11.1 (pH 7.4)
that had been saturated with 95% O2/5% CO2.
Ring segments were cut from each artery and mounted on stainless steel
hooks. Rings were placed at resting tension in organ baths containing
25 mL of buffer (37°C). The upper hook was connected to a force
displacement transducer, and isometric tension was displayed
continuously on a polygraph. Tension was increased to optimal tension
in a stepwise manner, to 0.5 g (basilar artery) and 2 g
(carotid artery) over 1 hour, and tension was periodically adjusted for
an additional 45 minutes for equilibration. During this period the
buffer was replaced at 15-minute intervals. All preparations had intact
endothelium as demonstrated by relaxation to
acetylcholine. In each experiment concentration-response curves to
histamine, phenylephrine, and KCl were obtained, and the
concentrations of these agents that produced 60% of the maximum
contractile response (EC60) were used in subsequent
studies. We have used these methods previously.27,28 All
procedures were performed in compliance with guidelines established by
the Institutional Animal Care and Use Committee.
Experimental Protocol
Circulating complexes of LDL-malonyldialdehyde have been
detected in plasma.29,30 Modified LDL, similar to that
oxidized in vitro, has been found in low concentrations in plasma in
humans (
5% of total plasma LDL).31 The low plasma
concentration of modified LDL probably is due to both plasma
antioxidants and removal by the liver of modified LDL.6
Therefore, in the present study vessels were exposed to a low
concentration of mildly ox-LDL (100 µg protein per milliliter of ring
chamber buffer) by means of a reaction (X/XO) that generates oxygen
radicals in amounts similar to those observed in vivo in several
pathophysiological conditions.7-9
After equilibration, endothelium-dependent relaxation was tested in all vessels with the use of acetylcholine, while the vessels were contracted with histamine, phenylephrine, or KCl. Changes in tension were measured after preincubation for 30 minutes with n-LDL or ox-LDL (100 µg protein per milliliter of buffer). In parallel experiments, we tested effects of ox-LDL in the presence of SOD, catalase, or DMTU added to the tubes before reactive oxygen species were generated by X/XO. After the concentration-response curve was obtained, vessels were washed twice with buffer and allowed to return to resting tension for at least 30 minutes.
To determine the effect of n-LDL and ox-LDL on relaxation, arterial rings were contracted with an EC60 dose of histamine (basilar and carotid arteries) or phenylephrine or KCl (carotid arteries). When stable contraction had developed, a concentration-response curve for relaxation was obtained by addition of acetylcholine or sodium nitroprusside. Similar to the procedure used in the first part of the experiment, after the concentration-response curve for relaxation was obtained, vessels were washed several times with buffer and allowed to return to resting tension for at least 30 minutes. In parallel experiments in other vascular rings we tested the effects of ox-LDL in the presence of SOD, catalase, or DMTU added to the tubes before reactive oxygen species were generated by X/XO. This experimental procedure was repeated for each vasodilator.
Statistical Analysis
Values are expressed as mean±SEM. A repeated measures ANOVA,
with Bonferroni adjustment for multiple comparisons, was used to
compare LDL with ox-LDL and ox-LDL with ox-LDL plus different reactive
oxygen species scavengers. Values of P<.05 were considered
significant.
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Results |
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Effects of n-LDL and ox-LDL on Responses to Histamine, Phenylephrine, and KCl
Compared with control vessels, preincubation with n-LDL did not alter dose-response curves obtained with histamine, acetylcholine, or nitroprusside in basilar or carotid arteries (data not shown).
Contraction of the basilar arteries to histamine was not altered by
ox-LDL (Fig 1
). In contrast, contractions
of the carotid arteries to histamine and phenylephrine were
impaired in the presence of ox-LDL (Fig 2). Impairment of contraction was
prevented by pretreatment of the LDL solution with either SOD,
catalase, or the hydroxyl radical scavenger DMTU before addition of
X/XO (Fig 2). These data suggest that ox-LDL impaired contraction in
the carotid but not basilar arteries and that alteration of LDL was
mediated by reactive oxygen species, perhaps the hydroxyl radical.
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P<.05, ox-LDL vs ox-LDL plus SOD, catalase, or
DMTU.
Additional experiments were performed in carotid arteries to determine
whether ox-LDL impairs contraction to the receptor-independent stimulus
KCl. There was significant impairment by ox-LDL of maximum contraction
to KCl (Fig 3).
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Effects of n-LDL and ox-LDL on
Endothelium-Dependent and
Endothelium-Independent Relaxation
n-LDL did not alter responses to acetylcholine in precontracted
basilar and carotid arteries (data not shown). Acetylcholine produced
similar relaxation in the basilar artery precontracted with histamine
in the presence of n-LDL or ox-LDL (Fig 4). In contrast, ox-LDL impaired
relaxation to acetylcholine in the carotid artery that was
precontracted with histamine or phenylephrine (Fig 5). Impaired relaxation was prevented by
pretreatment of LDL with either SOD, catalase, or the hydroxyl radical
scavenger DMTU (Fig 5). These results suggest that ox-LDL may impair
endothelium-dependent relaxation in carotid but not in
basilar arteries.
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To determine whether endothelium-independent relaxation
was altered after treatment of arteries with ox-LDL, we examined the
effects of ox-LDL on responses to sodium nitroprusside. Relaxation
induced by sodium nitroprusside was similar in the presence of n-LDL or
ox-LDL in histamine-precontracted carotid and basilar arteries (Fig 6). Relaxation induced by sodium
nitroprusside was impaired in the presence of ox-LDL in
phenylephrine-precontracted carotid arteries (Fig 7).
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Discussion |
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The major finding in this study is that a relatively brief exposure to low concentrations of LDL oxidized with X/XO impairs reactivity in carotid but not basilar arteries. Impairment of responses by ox-LDL in the carotid artery was prevented by pretreatment of LDL with SOD, catalase, or DMTU. These results suggest that intracranial arteries may be protected more effectively than extracranial arteries from injury mediated by ox-LDL. These findings may have implication for epidemiological,1 clinical,1,3 and histological studies1,2 which suggest that atherosclerosis occurs later or to a lesser extent in intracranial than extracranial vessels. Moreover, in atherosclerotic rabbits, endothelium-dependent relaxation has been reported to be impaired in aorta and carotid arteries but not basilar arteries in vitro,32 although another study reported that endothelium-dependent relaxation is impaired in basilar arteries of atherosclerotic rabbits.33
Because pretreatment of LDL with either catalase or SOD prevented impairment of vascular reactivity in carotid arteries by ox-LDL, both superoxide radical and hydrogen peroxide may be necessary for oxidation of LDL. These data are also consistent with the possibility that the radical immediately responsible for oxidation of LDL by X/XO was hydroxyl radical, generated by means of Haber-Weiss reaction from superoxide radical and hydrogen peroxide.34 This hypothesis is supported by the results of experiments in which the hydroxyl radical scavenger DMTU was used.
Generation of reactive oxygen species by the X/XO reaction produces mild oxidation of human LDL20-22 in a range that may be encountered in vivo.7-9 The X/XO reaction also produces hydrogen peroxide and hydroxyl radical.25,26 The latter is generated in vivo in the presence of trace amounts of iron or other transition heavy metals34,35 and can be enhanced by iron.36 Furthermore, hydroxyl radical may be formed in vivo by reaction of nitric oxide with superoxide anion, which may lead to LDL peroxidation.37 All of these compounds could contribute to oxidation of LDL and impairment of vascular responses. Alternatively, reactive oxygen species may produce oxidation of LDL, and their action may be additive. Thus, protection of the LDL solution by SOD and catalase before addition of X/XO may be due to reduction of the total concentration of reactive oxygen species–mediated injury, irrespective of different species responsible for these impairments.
There is substantial evidence that modification of LDL by reactive oxygen species occurs in vivo.5 Prevention of oxidative modification of LDL or minimization of its oxidative effects improves receptor-mediated endothelium-dependent relaxation of vessels from hypercholesterolemic rabbits.38,39
In the present study ox-LDL inhibited acetylcholine-induced relaxation in the carotid arteries but not in basilar arteries. A recent study40 reported that LDL modified by cigarette smoke and LDL oxidized by CuCl2 (ox-LDL) inhibits acetylcholine-induced relaxation in the basilar arteries. The difference between the present findings and the previous study may be related to different methods that were used to oxidize LDL or higher concentrations of LDL that were used in the previous study (2 mg protein per milliliter of ox-LDL and 0.5 mg protein per milliliter of LDL modified by cigarette smoke versus 100 µg protein per milliliter of LDL or ox-LDL in the present study).
Acetylcholine releases nitric oxide from endothelial cells, and nitroprusside relaxes vessels by release of nitric oxide as its active compound. The results obtained with sodium nitroprusside in vessels precontracted with histamine suggest that responses to nitric oxide were relatively preserved in both carotid and basilar arteries after brief exposure to low concentrations of LDL oxidized with X/XO reaction. We observed some impairment of the responses to sodium nitroprusside in the carotid arteries precontracted with phenylephrine in the presence of ox-LDL. The explanation for modest impairment of responses when vessels are precontracted with phenylephrine, but not histamine, is not clear.
Our data suggest that oxidized LDL may impair intracellular mechanisms that mediate contraction of the carotid arteries. This possibility is supported by experiments in which both receptor-mediated stimuli and KCl, a receptor-independent contractile stimulus, are used. Mechanisms that account for the difference between carotid and basilar arteries in relation to susceptibility to ox-LDL are not clear. Differences in susceptibility to contraction could be explained by differences in the content of antioxidants in membranes or by the different lipid composition in endothelial cells of basilar arteries, which may trap reactive oxygen species. Because contraction induced by KCl was impaired at high doses, there may be a direct effect of ox-LDL on smooth muscle cells.
It therefore appears that there are two different pathophysiological mechanisms that affect the carotid artery, which are triggered by X/XO-modified LDL. Modified LDL produces endothelial dysfunction and impairment of contraction of vascular muscle. It is difficult to compare results of studies12-16 of effects on vasoreactivity of n-LDL and ox-LDL. Oxidation of LDL may be augmented by lengthy procedures to isolate LDL, dialysis of LDL, and the absence of adequate measures to protect against oxidation of both lipid and protein. Hence, without adequate protection of the oxidative status of LDL preparations,41 effects that are observed in response to n-LDL may not be a response to n-LDL, because the degree of LDL oxidation may be greater than expected. Aerated organ chambers to which LDL was added also represent a highly oxidizing environment. In the present study further modifications of all LDL samples (n-LDL and ox-LDL) was prevented by adding the antioxidant butylated hydroxytoluene to the tubes of reaction, thus preventing subsequent chain reactions of peroxidation.20 We previously reported in LDL oxidized by X/XO an increased mobility on agarose electrophoresis of LDL apolipoprotein-B100 due to enhanced amino acid negative charge.21,22 This alteration may confer to ox-LDL an ability to interact with vascular cells.
In conclusion, ox-LDL impairs contraction and endothelium-dependent relaxation in the carotid but not in the basilar artery. We speculate that differences in vasoreactivity between basilar and carotid arteries after exposure to ox-LDL may be due in part to a different content of scavengers of oxygen radicals. Thus, intracranial arteries may be relatively resistant to mildly ox-LDL.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health grants NS-24621, HL-16066, and HL-14388; research funds from the Veterans Administration; and grant 94.00.157 (Progetto Finalizzato Prevenzione e Controllo Fattori di Malattia) from Consiglio Nazionale delle Ricerche, Rome, Italy. The authors wish to thank Professor Mario Mancini (Federico II University of Naples) and Dr David Chappell (University of Iowa) for assistance in preparation of the LDL and manuscript. The authors wish to thank Dr David Rios (University of Iowa) for his help during the study. Dr Faraci is an Established Investigator of the American Heart Association. This study was conducted while Dr Napoli was a visiting scientist at the University of Iowa.
Received April 7, 1997; revision received August 5, 1997; accepted August 19, 1997.
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F. P. D'Armiento, A. Bianchi, F. de Nigris, D. M. Capuzzi, M. R. D'Armiento, G. Crimi, P. Abete, W. Palinski, M. Condorelli, C. Napoli, et al. Age-Related Effects on Atherogenesis and Scavenger Enzymes of Intracranial and Extracranial Arteries in Men Without Classic Risk Factors for Atherosclerosis Editorial Comment Stroke, November 1, 2001; 32(11): 2472 - 2480. [Abstract] [Full Text] [PDF]
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 C. Napoli and W. Palinski Maternal hypercholesterolemia during pregnancy influences the later devolopment of atherosclerosis: clinical and pathogenic implications Eur. Heart J., January 1, 2001; 22(1): 4 - 9. [PDF]
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 C. NAPOLI, O. QUEHENBERGER, F. DE NIGRIS, P. ABETE, C. K. GLASS, and W. PALINSKI Mildly oxidized low density lipoprotein activates multiple apoptotic signaling pathways in human coronary cells FASEB J, October 1, 2000; 14(13): 1996 - 2007. [Abstract] [Full Text]
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H. Xie, J. A. Bevan, and W. G. Mayhan Oxidized Low-Density Lipoprotein Enhances Myogenic Tone in the Rabbit Posterior Cerebral Artery Through the Release of Endothelin-1 • Editorial Comment Stroke, November 1, 1999; 30 (11): 2423 - 2430. [Abstract] [Full Text] [PDF]
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C. Napoli, S. Salomone, T. Godfraind, W. Palinski, D. M. Capuzzi, G. Palumbo, F. P. D'Armiento, R. Donzelli, F. de Nigris, R. L. Capizzi, et al. 1,4-Dihydropyridine Calcium Channel Blockers Inhibit Plasma and LDL Oxidation and Formation of Oxidation-Specific Epitopes in the Arterial Wall and Prolong Survival in Stroke-Prone Spontaneously Hypertensive Rats • Editorial Comment Stroke, September 1, 1999; 30(9): 1907 - 1915. [Abstract] [Full Text] [PDF]
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C. Napoli, J. L. Witztum, F. de Nigris, G. Palumbo, F. P. D'Armiento, and W. Palinski Intracranial Arteries of Human Fetuses Are More Resistant to Hypercholesterolemia-Induced Fatty Streak Formation Than Extracranial Arteries Circulation, April 20, 1999; 99(15): 2003 - 2010. [Abstract] [Full Text] [PDF]
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 K. G. Lamping, D. W. Nuno, D. A. Chappell, and F. M. Faraci Agonist-specific impairment of coronary vascular function in genetically altered, hyperlipidemic mice Am J Physiol Regulatory Integrative Comp Physiol, April 1, 1999; 276(4): R1023 - R1029. [Abstract] [Full Text] [PDF]
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