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Hypertension. 2000;36:436-441

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(Hypertension. 2000;36:436.)
© 2000 American Heart Association, Inc.

Scientific Contributions

Probucol Reduces Oxysterol Formation in Hypertensive Rabbits

Howard N. Hodis; Sam Hashimoto; Wendy J. Mack; Alex Sevanian

From the University of Southern California School of Medicine, Atherosclerosis Research Unit, Division of Cardiology (H.N.H., S.M., W.J.M., A.S.), and Department of Preventive Medicine (H.N.H., W.J.M.), Los Angeles; and the University of Southern California School of Pharmacy, Department of Molecular Pharmacology and Toxicology (H.N.H., A.S.), Los Angeles.

Correspondence to Howard N. Hodis, MD, University of Southern California School of Medicine, Atherosclerosis Research Unit, Division of Cardiology, 2250 Alcazar St, CSC 132, Los Angeles, CA 90033. E-mail watcher{at}hsc.usc.edu


*    Abstract
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 *Abstract
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Abstract—The role of lipid peroxidation during the pathogenesis of atherosclerosis has been described through numerous studies and has provided compelling evidence for free radical–mediated processes that link hypertension with atherosclerosis. However, there remains only limited information concerning peroxidative processes in hypertension and their modulation by antioxidants. In the present study, the formation of cholesterol oxidation products was used as a measure of in vivo lipid peroxidation after hypertension induced by coarctation of the aorta in New Zealand White rabbits. The rabbits were fed a standard chow diet devoid of cholesterol or cholesterol oxidation products such that the measured cholesterol oxides in the plasma and aortic tissues would most plausibly arise from endogenous oxidation of cholesterol. After 12 weeks of hypertension, all of the measured cholesterol oxides increased significantly over baseline levels in the surgically coarctated animals; however, this increase was significantly less in hypertensive probucol-treated animals. Similarly, the cholesterol oxide content of aortic tissue from the surgically coarctated animals was significantly greater than that found in normotensive control aortas, and probucol treatment significantly reduced the increase in cholesterol oxide content of aortic tissue relative to that of hypertensive animals not receiving the antioxidant. These findings in hypertensive animals suggest that cholesterol oxidation products measured in plasma and aortic tissue can be derived from endogenous free radical activity and that this activity is enhanced under specific pathological conditions.


Key Words: lipids • hypertension, experimental • free radicals • cholesterol • antioxidants


*    Introduction
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 *Introduction
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Although hypertension is clearly a risk factor for atherosclerosis,1 the mechanism by which hypertension potentiates atherosclerosis remains to be elucidated.2 3 Evidence for free radical–mediated processes in the hypertensive arterial wall may provide a link between hypertension and atherosclerosis.4 5 6 The involvement of lipid peroxidation during the pathogenesis of atherosclerosis has been extensively studied.7 8 9 10 However, only limited information concerning peroxidative processes in hypertension and their modification by antioxidants is available.11 12 13 14 15 16

Because cholesterol oxides (ChOx) have been ubiquitously identified in human plasma and atheromatous lesions, their involvement in the atherogenic process is receiving increasing scrutiny.17 18 19 20 21 ChOx may influence the atherogenic process in several ways, since they are cytotoxic toward the cellular components of the arterial wall,22 23 24 increase vascular permeability,25 promote cholesterol ester formation and accumulation,26 27 28 inhibit prostaglandin I2 synthesis by endothelial cells,29 and perturb cholesterol biosynthesis.30 31

Previously, we and others have shown that cholesterol feeding of New Zealand White rabbits leads to increased plasma and aortic wall cholesterol oxide content,7 32 which is reduced with the antioxidant agent probucol.8 Information concerning the formation and accumulation of ChOx in plasma and arterial wall tissue as a result of hypertension has, on the other hand, not been described. Since it is well established that a number of ChOx are formed through free radical–mediated peroxidation of lipids containing cholesterol,22 measurement of these ChOx in lipids isolated from plasma and tissues could serve as a marker for free radical reactions in vivo. On the basis of accumulating evidence for the importance of ChOx in the atherosclerosis process and possible linkage between hypertension and atherosclerosis through free radical–mediated processes, we conducted this study to determine if ChOx were formed after coarctation-induced hypertension in New Zealand White rabbits and if their formation could be altered by antioxidant treatment with probucol. This model provides an approach to measuring ChOx under disease conditions that do not involve cholesterol feeding and the potentially confounding contributions of dietary ChOx.


*    Methods
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Experimental Procedure
Twenty male New Zealand White rabbits, purchased from a local breeder, were randomly divided into 3 experimental groups: hypertensive with standard rabbit chow diet (H), hypertensive with standard rabbit chow diet supplemented with probucol (HP), and sham-operated normotensive controls with standard rabbit chow diet (C). Under general anesthesia (2 mL ketamine [100 mg/mL]-xylazine [20 mg/mL]), hypertension was induced in 15 animals by partial ligation of the abdominal aorta immediately above the renal arteries with the use of sterile operative techniques.33 After the surgical procedure, 8 animals (weight, 2.76±0.32 kg) (designated as the H group) were continued on standard rabbit chow diet (Purina Mills, Inc); 7 animals (weight, 2.81±0.20 kg) (designated as the HP group) received the same standard rabbit chow supplemented with 1% probucol for 12 weeks. Five animals (weight, 2.79±0.22 kg) (designated as the C group) underwent the same surgical procedure as the hypertensive groups except for ligation of the aorta and were continued on standard rabbit chow diet for the same time period. All procedures followed were in accordance with institutional guidelines.

Supplementation of the standard rabbit chow with probucol was initiated in the HP animals the day of the surgical procedure. These diets were prepared by dissolving probucol in freshly distilled ether that was evenly sprayed as a fine mist into the rabbit chow and air-dried at room temperature (22°C) before being fed to the rabbits. Diets were prepared fresh every 3 days, and any food remaining in the feeding bins was replaced with fresh food. Three rabbits from the H group and 2 animals from the HP group died.

Blood Pressure Determinations
Indirect systolic blood pressure measurements were obtained on all animals before the surgical procedure by the ear capsule method of Grant and Rothschild, as described previously,34 and after surgery on a weekly basis until the end of the study. Each blood pressure analysis is the result of an average of >=6 successive determinations of systolic pressure performed on a given rabbit. The results were averaged to obtain the mean and standard error for each treatment group.

Determination of Plasma ChOx
Details of the analytical procedures are described elsewhere.7 In brief, blood was drawn from the central ear artery into EDTA-containing tubes (1.5 mg EDTA/mL of blood) after a 12-hour fast. Plasma was immediately prepared by centrifugation of the blood at 3000 rpm at 5°C. Samples were stored in the dark under argon at -70°C until analysis. The lipids were extracted by a modified Bligh-Dyer procedure7 and applied to "Diol" solid-phase extraction columns (VWR Scientific). The cholesterol/ChOx was collected, hydrolyzed by cold alkaline saponification, derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (Pierce), and analyzed by gas chromatography. Gas chromatography was performed with the use of a Shimadzu GC-14 fitted with a DB-1 capillary column (30 mx0.25-mm id, 0.25-µm film thickness, J&W Scientific Inc). All analyses were performed under anaerobic conditions in freshly prepared solvents containing 0.01% BHT.

Determination of Aortic Tissue ChOx
Aortas were dissected from the animals and analyzed as described previously.7 In brief, the aorta from the aortic valve to the surgical coarctation was isolated, stripped of adventitial tissue, washed with cold PBS containing 1 mg/mL of EDTA, and weighed. The aortic tissue was immediately frozen at -70°C and stored in the dark under argon until analysis. The aortic tissue was suspended in PBS containing EDTA and homogenized under a stream of nitrogen with a Tekmar homogenizer in the extraction solvent of chloroform/methanol (2:1 vol/vol) containing 0.01% BHT. Total lipids were collected from the homogenization solvent, evaporated under nitrogen, redissolved in toluene/ethyl acetate, and applied to "Diol" solid-phase extraction columns. The cholesterol/cholesterol oxide fraction obtained from the extraction column was then hydrolyzed by cold alkaline saponification, derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide, and analyzed by gas chromatography as described above.

Independent standards containing pure cholesterol with and without probucol were processed and analyzed under the same conditions as the biological specimens to determine extent of ChOx formation during the preparative and analytical process. The cholesterol concentrations used were equivalent to the mean plasma cholesterol levels.

ChOx content of the rabbit chow was determined with and without probucol supplementation on the last day (third day) of the feeding period. The chow ({approx}1 g) was disrupted by homogenization in the chloroform/methanol extraction solvent and the lipid extract processed as described above for cholesterol/ChOx determinations.

Statistics
Mean differences between experimental groups were tested for statistical significance by performing pairwise Student’s t tests for independent samples. To correct for multiple hypothesis testing (3 pairwise comparisons per variable), a Bonferroni adjustment was used so that the {alpha}-level for any t test was 0.017 (0.05/3). Thus, group mean differences associated with a value of P<0.017 were considered statistically significant. All statistical testing was 2-sided. Values in tables are presented as mean (SEM).


*    Results
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Body weights of the rabbits from the 3 experimental groups were not statistically different at the time of autopsy (H: 3.91±0.41 kg, HP: 3.93±0.20 kg, C: 3.96±0.22 kg). Baseline blood pressures in all 3 experimental groups were equal (Table 1). After coarctation of aortas in the H and HP groups, a steady rise in blood pressure was observed that became significantly increased from baseline in both surgical groups by the second week. There were no significant differences in blood pressure at the end of the study between the 2 experimental hypertensive groups or at any time point during the 12-week study period. Blood pressure in the C group remained unchanged throughout the study period (Table 1).


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  		Table 1. Baseline and Week 12 Blood Pressure (Systolic mm Hg)

At baseline, there were no significant differences in plasma total cholesterol levels and plasma ChOx levels of 7-ketocholesterol, cholest-5-ene-3ß,7ß-diol, 5,6{alpha}-epoxy-5{alpha}-cholestan-3{alpha}-ol, and 5{alpha}-cholestane-3ß,5,6ß-triol between the 2 hypertensive groups of animals (Table 2). These values, as expected, were comparable to those from normotensive control animals (Table 2). After 12 weeks of hypertension, all of the measured ChOx in plasma increased significantly over their respective baseline levels in both groups of surgically coarctated animals (Table 2). However, the increase in the plasma ChOx levels in the hypertensive probucol-treated animals was significantly less than that of the hypertensive animals not receiving antioxidant therapy (Table 2). Although probucol reduced the rise in each ChOx by {approx}50%, it did not completely inhibit formation of these sterol products. The ChOx levels remained significantly greater than those at baseline as well as greater than the control values (Table 2). On the other hand, there were no significant differences in plasma total cholesterol levels either within each hypertensive group of animals, between the 2 hypertensive groups, or relative to the normotensive control group (Table 2).


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  		Table 2. Plasma Total Cholesterol and Oxysterol Levels (mg/dL)

After 12 weeks of hypertension, the ChOx content of aortic tissue from the surgically coarctated groups of animals was significantly greater than that found in normotensive control aorta (Table 3). Probucol treatment during the 12-week period of hypertension significantly reduced the increase in ChOx content of aortic tissue relative to that of hypertensive animals not receiving the antioxidant (Table 3). The ChOx identified in aortic tissue were the same as those found in plasma, being largely comprised of 7-ketocholesterol, cholest-5-ene-3ß,7ß-diol, 5,6{alpha}-epoxy-5{alpha}-cholestan-3{alpha}-ol, and 5{alpha}-cholestane-3ß,5,6ß-triol. As in plasma, probucol significantly but not completely reduced the increase of aortic tissue ChOx levels in hypertension to normotensive control levels (Table 3). As a percentage of the total cholesterol content of aortic tissue, hypertension induced an {approx}4-fold increase of ChOx, whereas addition of probucol reduced this increase approximately by half (Table 3). Relative to the cholesterol content of aortic tissue from normotensive control animals, aortic tissue content of cholesterol from both hypertensive groups of animals was not significantly different (Table 3). Probucol apparently had no effect on the cholesterol content of aortic tissue relative to the hypertensive group of animals not receiving this agent (Table 3).


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  		Table 3. Aortic Tissue Cholesterol and Oxysterol Content at Week 12 (ng/mg)

Analysis of the feed revealed that there were no detectable levels of ChOx in the rabbit chow in standard chow prepared with or without probucol. Indeed, only traces of cholesterol were present among a series of unidentified sterols that were probably of plant origin.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
This study demonstrated that coarctation-induced hypertension in New Zealand White rabbits leads to a rise in plasma ChOx levels without alteration of the plasma total cholesterol level. Furthermore, ChOx content of aortic wall tissue from hypertensive animals was significantly greater than in normotensive control rabbits. The ChOx described represent the major isolable species that were consistently and reliably measured in all animals. Other minor ChOx were found in these samples; however, the levels were often at the limits of detection or were entirely absent in many samples. These results implicate the formation of free radical–mediated processes during hypertension that are represented by ChOx formation. Since there was no cholesterol and cholesterol oxidation products in the food consumed by these rabbits, the contribution of dietary ChOx to the levels measured in plasma or tissue can be excluded. Thus, an oxidative origin for the ChOx measured in the animals becomes more plausible. In accord with this conclusion is the finding that the antioxidant agent probucol partially but significantly inhibited the rise in plasma cholesterol oxide levels and the ChOx content of aortic wall tissue. Probucol has known antioxidant activity both in vitro and in vivo and has been shown to decrease cholesterol oxidation products in hypercholesterolemia.8 Probucol had a small and nonsignificant effect in terms of cholesterol lowering in this study and only in hypertensive rabbits. This minor effect may be due to the already low cholesterol levels; however, it does not directly demonstrate the extent to which probucol was absorbed in these animals.

Free radical–mediated autoxidation of cholesterol and partial inhibition by antioxidant administration provide evidence for free radical–mediated processes within the hypertensive aortic wall in New Zealand White rabbits. This process may form a link between hypertension and atherosclerosis. Measurement of ChOx as a reflection of lipid peroxidation in vivo provides a reliable means for determining whether increased free radical activity has taken place. There is strong evidence linking the formation of cholesterol oxidation products with lipid peroxidation processes, both in vitro35 and in vivo.36 Although other lipid peroxidation products have been measured in plasma and tissues, many of which appear to be reliable markers of free radical events in vivo, the mechanism of formation of the ChOx identified in this study are well known to be oxyradical mediated. Moreover, because of the relative stability of many ChOx, their detection in biological materials, particularly serum lipoproteins, has important advantages over measurements of other lipid peroxidation products, many of which are labile. For example, malondialdehyde, peroxides, conjugated dienes, and isoprostanes are difficult to measure because of their instability in biological matrixes and/or presence of numerous interfering substances.

Several reports have implicated the role of lipid peroxidation in hypertension,4 5 6 and specific oxidative changes to lipids during hypertension have been previously reported, particularly in the context of inactivation and sequestration of nitric oxide (NO). For example, amelioration by vitamin E of lead-induced hypertension, tissue damage, and reduced NO production also supports the role of increased reactive oxygen species.37 It has been suggested that free radical–mediated processes, which include cholesterol as a target for oxidative modification, play an important role in the pathogenesis of atherosclerosis.21 Cholesterol oxidation products may be one of the agents responsible not only for vascular injury as discussed above but may also be responsible for the inhibition of NO production by the vascular endothelium.38 There is evidence that the lipoxygenase pathway is a mediator of angiotensin II, implicating a role for humoral factors such as angiotensin II in mediating oxidative stress responses in the vessel wall.39 Thus, activation of lipoxygenase may be a possible mechanism contributing to formation of lipid peroxides and oxysterols. Because probucol was able to inhibit cholesterol oxidation in hypertensive rabbits, this finding provides further evidence for continued studies on peroxidation-mediated vascular effects in hypertensive animals.

The role, if any, that oxidized cholesterol may play in hypertension is unknown at the present time. However, oxygen-derived free radicals have been suggested to participate in the pathogenesis of hypertensive vascular disease by increasing vascular permeability and contractility as well as by inducing cellular injury, the interpretation of which is based on the inhibitory effects of free radical scavengers.11 12 13 40 The biological effects seen with oxygen-derived free radicals noted above also occur with ChOx.

Hypertension enhances atheromatous lesions in the presence of hypercholesterolemia in experimental animals.2 3 Elevated levels of cholesterol in plasma and vascular tissue may provide a higher concentration of cholesterol substrate susceptible to oxygen-derived free radicals induced by hypertension. Although the pathogenesis of free radicals in hypertension is incompletely understood, many sources are possible, including free radical generation by polymorphonuclear leukocytes and enhanced production of radical species and oxidants by endothelium and smooth muscle cells.5 11 41 Cellular oxidative modification of cholesterol is thought to involve endothelial- and macrophage-derived reactive oxygen species.36 42 43 Hypertension, which increases arterial wall thickness and oxygen diffusion, produces medial hypoxia and steep oxygen tension gradients within the wall.44 Recently, compelling evidence was presented that hypoxic areas do indeed exist, particularly in the thickened arterial wall.45 Intermittent hypoxia and steep oxygen tension gradients may lead to oxyradical formation by disrupting oxygen metabolism and accelerating free radical formation through partially reduced oxygen products. This disruption of oxygen diffusion and associated reactive oxygen species formation may be related to the induction of antioxidant enzymes in the aortic wall of hypertensive rabbits.46

In this study, it was determined that the feed with or without probucol contained undetectable levels of ChOx. Therefore, it seems unlikely that exogenous sources of these products accounted for differences between the experimental groups of animals. These findings in hypertensive animals support the observations of others47 that ChOx measured in plasma and aortic tissue can be derived from endogenous free radical activity and that this activity is enhanced under specific pathological conditions.

Finally, the significant effect of partially inhibiting cholesterol oxide formation with antioxidant administration during induction of hypertension raises the question of whether antioxidants may be used to prevent hypertensive arterial wall changes.48 We recently reported that injection of oxysterols49 increased the amount of oxysterol deposition in the aortic wall and when combined with cholesterol feeding also increased the deposition of cholesterol and fatty streak formation.50 Oxysterol content in the aortic arch and abdominal aorta increased; however, the amount of oxysterols and extent of cholesterol deposition was much lower in the abdominal aorta. These differences indicate that factors such as shear force or humoral responses may influence the extent of oxidative stress, promoting the formation of lipid peroxidation products in areas of predilection. Antioxidants have proven ameliorating effects in atherosclerosis arterial wall changes in experimental animals in which probucol has been shown to decrease the arterial wall content of ChOx along with the cholesterol content during induction of hypercholesterolemia.8 Antioxidant procyanidins also have been shown to prevent cholesterol deposition and oxysterol production in hypercholesterolemic rabbits independent of cholesterol-lowering effects, suggesting that modulation of oxidative stress has a significant impact on factors that influence vascular function.51 52 Further studies to determine the utility of antioxidants in altering the pathogenesis of hypertensive arterial wall changes will be required.


*    Acknowledgments
 
This work was supported by the Wright Foundation Research Award from the University of Southern California School of Medicine, by grant ES03466 from the National Institutes of Health, NATO International Collaborative grant CRG 920513, and a research grant from Merrill Dow Pharmaceuticals.

Received October 15, 1999; first decision January 4, 2000; accepted March 31, 2000.


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 *References
 

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