Published online before print June 16, 2005, doi: 10.1161/01.ATV.0000174125.89058.b6
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© 2005 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Processes Involved in the Site-Specific Effect of Probucol on Atherosclerosis in Apolipoprotein E Gene Knockout Mice
From Centre for Vascular Research (K.C., K.B., F.Y.P., B.J.W., S.R.T., R.S.), University of New South Wales, Sydney, Australia; Heart Research Institute (S.B.L., J.Y.H.), Sydney, Australia; University of Western Australia (K.D.C., T.A.M.), School of Medicine and Pharmacology, Perth, Western Australia, Australia.
Correspondence to Prof Roland Stocker, Centre for Vascular Research, School of Medical Sciences, Faculty of Medicine, University of New South Wales, UNSW Sydney, NSW 2052, Australia. E-mail r.stocker{at}unsw.edu.au
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Abstract |
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Objective— To elucidate processes by which the antioxidant probucol increases lesion size at the aortic sinus and decreases atherosclerosis at more distal sites in apolipoprotein E–deficient (apoE–/–) mice.
Methods and Results— Male apoE–/– mice were fed high-fat chow with 1% (w/w) probucol or without (controls) for 6 months, before aortic sinus, arch, and descending aorta were analyzed separately for lesion size and composition. Compared with control, probucol significantly increased lesion size by 33% at the sinus, but it inhibited atherosclerosis at the descending aorta by 94%. Sites where atherosclerosis was inhibited contained substantially fewer macrophages, less lipids (cholesterol and cholesteryl esters), and endogenous antioxidant (
-tocopherol), but not oxidized lipids, and the extent to which probucol metabolism occurred was increased. Compared with control, aortic sinus lesions of probucol mice contained a substantially increased content of extracellular matrix, but decreased total cell and macrophage density, comparable levels of lipids and -tocopherol, and decreased concentrations of oxidized lipids (cholesteryl ester hydroperoxides, F2-isoprostanes, and 7-ketocholesterol).
Conclusions— Probucol affects atherosclerosis in apoE–/– mice independent of the accumulation of arterial lipid oxidation products, thereby dissociating the 2 processes. Rather, probucol exerts antiinflammatory activity by decreasing accumulation of macrophages in lesions, and it promotes a more stable lesion composition at the aortic sinus.
We investigated how the antioxidant probucol increases lesion size at the sinus and decreases atherosclerosis at distal sites in apolipoprotein E-deficient mice. Probucol affected atherosclerosis independent of arterial lipid oxidation. Rather, probucol decreased accumulation of macrophages in lesions, and it promoted a more stable lesion composition at the aortic sinus.
Key Words: antioxidants • atherosclerosis • collagen • free radicals • inflammation
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Introduction |
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Atherosclerosis represents a state of heightened oxidative stress characterized by lipid and protein oxidation in the vascular wall.1 The oxidative modification hypothesis of atherosclerosis predicts that low-density lipoprotein (LDL) oxidation is an early event in and that oxidized LDL contributes to atherogenesis.2 Oxidized LDL supports foam cell formation in vitro and other potentially pro-atherogenic activities, the lipid in human lesions is oxidized and contains oxidized LDL, and several different antioxidants inhibit atherosclerosis in animals.1 In addition to LDL oxidation, other relevant oxidative events include the production of reactive oxygen and nitrogen species by vascular cells,3 and oxidative modifications contributing to important clinical manifestations of coronary artery disease such as endothelial dysfunction and plaque disruption.1
However, despite abundant data, fundamental problems remain with implicating oxidative modification as a requisite cause for atherosclerosis.1 These include the poor performance of antioxidants in limiting atherosclerosis or cardiovascular events from it,4 and observations in animals that suggest dissociation between atherosclerosis and lipoprotein lipid oxidation.5–9 To reconcile these discrepancies, the "oxidative response to inflammation" model of atherosclerosis1 considers inflammation as a primary process of atherosclerosis, and oxidative stress as an event secondary to inflammation. However, this model too raises several important questions, including how antioxidants like probucol, that consistently inhibit atherosclerosis and related disorders, actually work.
Probucol is a phenolic antioxidant and rarely used cholesterol-lowering drug that attenuates atherogenesis in animals10 and humans,11 and that protects human coronary arteries from restenosis.12 In animal models of atherosclerosis, probucol alters the cellular composition and proliferation,13,14 and inhibits coronary heart disease and death.15,16 It also prevents intimal thickening after balloon injury independent of cholesterol-lowering and inhibition of lipoprotein lipid oxidation, and instead promotes functional re-endothelialization and inhibits vascular smooth muscle cell proliferation via induction of heme oxygenase-1.17,18
Strikingly, in apolipoprotein E–deficient (apoE–/–) mice, probucol increases lesion size at the aortic sinus19 while at the same time it strongly inhibits atherosclerosis in the descending aorta.6 In the present study, we used this model to elucidate processes by which probucol exerts this site-specific effect.
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Methods |
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ApoE–/– Mice
Male apoE–/– mice originally purchased from Jackson Laboratories (Bar Harbor, Me) were used at 8 to 10 weeks of age and then fed for 24 weeks ad libitum a high-fat diet containing 21.2 (w/w) fat and 0.15% (w/w) cholesterol (specifications of the Harlan Teklad diet TD88137), without (controls, 103 mice), or with probucol (1% w/w, 87 mice).6 The local animal ethics committee approved the study.
Aortic Sampling for Biochemical Analyses
Procedures were performed as described.6,20 For biochemistry (86 and 70 animals for control and probucol, respectively), hearts and aortas past the femoral bifurcation were excised, cleaned, placed immediately in ice-cold buffer containing protease inhibitors and antibiotics, and stored at –80°C. Tissues were separated into 2 groups, one for F2-isoprostanes and arachidonic acid determination (n=10 for each, control and probucol) and the other for total cholesterol, nonesterified cholesterol (NEC), 7-ketocholesterol (7KC), cholesteryl esters (CE) (defined as the sum of C18:2 plus cholesterylarachidonate, C20:4), CE-OOH, -tocopherol, and probucol and its metabolites (n=76 and 60 for control and probucol, respectively), with analyses as described in detail in supplemental material (please see http://atvb/ahajournals.org).
Histology
For intimal lesion assessment, perfusion-fixed hearts and aortas (n=17 for each, controls and probucol) were subjected to blinded morphometry at the sinus, arch, and descending thoracic and abdominal aorta,6 as described in detail in supplemental material. For total cell numbers, macrophages, and collagen content, sections were taken 200 µm from the first appearance of the leaflets (aortic sinus) and 100 µm distal from the branch point of the third pair of intracostal arteries (thoracic aorta) and the celiac artery (abdominal aorta), and processed as described in detail in supplemental material.
Real-Time Reverse-Transcription Polymerase Chain Reaction
For reverse-transcription polymerase chain reaction analyses, animals were perfused with cold phosphate-buffered saline and aortas and hearts (n=20 for each control and probucol-treated animals) excised, immediately transferred into RNAlater (Ambion), stored for 24 hours at 4°C, and then stored at –80°C. Aortic sinus and thoracic/abdominal aortas were prepared as described and tissues from 10 animals each were pooled to obtain 2 pools per treatment per aortic site. Total RNA was isolated, cDNA prepared, and reverse-transcription polymerase chain reaction performed as described in detail in supplemental material.
Statistics
Results are shown as means±SEM. The effect of probucol on lesion size was analyzed by the Mann-Whitney U test. Biochemical parameters were compared using a 1-way ANOVA or Mann-Whitney U test. For total cell numbers, macrophages, and collagen content, differences between means were evaluated using the Student t test. Statistical significance was accepted at P<0.05.
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Results |
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Site-Specific Effect of Probucol on Atherosclerosis
In apoE–/– mice, probucol affects atherosclerosis nonuniformly.6 The results of the present study, using a large number of animals, confirmed this earlier observation (Figure I, available online at http://atvb.ahajournals.org). Probucol significantly increased lesion size by 33% at the sinus (Figure 1; P<0.01) (Table 1), whereas it visibly inhibited atherosclerosis in other parts of the aorta, including the carotid and femoral arteries (Figure I). Probucol increasingly inhibited disease along the aortic tree, with 36% inhibition at the arch (not significant) and 94% inhibition at the descending aorta (P<0.0001) (Figure 1 and Table 1).
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). Results show mean±SEM for 17 mice per group and for each site. *Significantly different from corresponding control value (P<0.05).
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Effect of Probucol on Nonoxidized Lipids and Lipid-Soluble Antioxidants
The ability of probucol to simultaneously promote and inhibit atherosclerosis provides an experimental model to directly relate the extent of lipoprotein lipid oxidation and atherogenesis in different aortic segments of the same animal. To do this, we first determined the concentrations of the nonoxidized lipids, NEC and CE, and the antioxidant -tocopherol as measures of lipoprotein lipid accumulation. For control and probucol-treated animals, lesions at the sinus contained more NEC per wet weight (Table I, available online at http://atvb.ahajournals.org) or protein (Table 1) than respective lesions at the arch and thoracic/abdominal aorta. Similarly, in control animals there was more CE per wet weight in the aortic sinus than thoracic/abdominal aorta. This reflects the relatively larger and more mature lesions at proximal than distal sites in apoE–/– mice.21 In contrast, the protein-standardized contents of C18:2, C20:4, and -tocopherol were not different at the 3 sites in control animals, whereas probucol significantly decreased the tissue content of CE and the vitamin, independent of whether results were expressed per wet weight or protein. Figure 2 is a graphic representation of the protein-standardized results, with data from probucol-treated mice expressed relative to that of control animals for each of the 3 sites. Compared with controls, probucol decreased the concentrations of NEC (Figure 2A), CE (Figure 2B), and -tocopherol (Figure 2C) in the arch and descending aorta, in parallel with inhibition of disease (Figure 1). However, probucol did not increase the content of NEC (Figure 2A), CE (Figure 2B), and -tocopherol (Figure 2C) at the sinus.
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Effect of Probucol on Lipid Oxidation in Atherosclerotic Lesions at Different Sites
We used 3 separate measures to assess lipid oxidation, ie, CE-OOH, F2-isoprostanes, and 7KC. CE-OOH represents the extent of lipoprotein lipid oxidation,20 F2-isoprostanes are a general marker of lipid oxidation,22 and 7KC is the most abundant oxysterol in atherosclerotic lesions.23 CE-OOH was the most abundant of these markers of lipid oxidation (Table I and Table 1). In control and probucol-treated mice, 7KC was not different at different sites, irrespective of how data were expressed. In contrast, protein- and parent lipid-standardized concentrations of CE-OOH and F2-isoprostanes were decreased at the thoracic/abdominal site compared with aortic sinus (Table 1). Figure 3 compares the parent lipid-standardized content of oxidized lipids at the 3 sites in control versus probucol-treated mice. At the sinus, where probucol increased lesion size (Figure 1), the drug decreased the concentrations of CE-OOH (Figure 3A), F2-isoprostanes (Figure 3B), and 7KC (Figure 3C), and this reached statistical significance in the case of F2-isoprostanes and 7KC. In contrast, probucol significantly increased CE-OOH and F2-isoprostanes at the descending aorta where the drug almost completely prevented atherosclerosis. We expressed all 3 parameters of lipid oxidation relative to the respective parent molecule (ie, CE for CE-OOH, arachidonate for F2-isoprostanes, and total cholesterol for 7KC) to distinguish lipid oxidation from lipid load, because the latter was affected significantly by probucol (Figure 2A and 2B). However, even when the lipid oxidation parameters were expressed per wet weight (Table I) or protein (not shown), their concentrations did not reflect the effect of probucol on lesion development.
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Tissue Levels of Probucol and Probucol Metabolites
We next assessed whether the site-specific effect on atherosclerosis was related to the drug concentration in the vessel wall. As probucol is metabolized,24 we also determined its known metabolite probucol bisphenol and diphenoquinone. Both probucol and the total amount of the drug were significantly lower in the descending aorta than the sinus (Table 1 and Table I). This is not surprising, given that probucol is transported in lipoproteins, so that the results reflected the extent of lipoprotein infiltration at these sites. Consistent with this, the amount of probucol was no longer different for the different sites, when the drug concentration was standardized to NEC plus CE (Figure 4A), NEC, or CE (data not shown) rather than protein (Table 1). In contrast, the concentration of probucol metabolites varied less at the 3 aortic sites, whether expressed per wet weight (Table I), protein (Table 1), or lipid-adjusted (not shown). When expressed relative to parent drug, the metabolites were significantly increased and accounted for nearly one-third of the drug at the descending aorta where atherosclerosis was inhibited compared with the aortic sinus where lesion size was increased (Figure 4B).
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Significantly different from corresponding sinus and arch value, respectively (P<0.05).
Histological Assessment of Aortic Sinus Lesions
Because neither differences in lipid accumulation nor the extent of lipid oxidation explained why lesions at the aortic sinus were larger in probucol-treated than control mice, we next determined the cellular composition at different sites. At the sinus, total cell numbers were similar in control and probucol-treated animals, so that probucol significantly decreased the number of cells per lesion area (Table 2). Similarly, the lesion area covered by macrophages was similar in control and probucol-treated animals, a finding confirmed by the lesion content of mRNA of F4/80 antigen, a specific marker for macrophages.25 As a consequence, probucol significantly decreased the percentage of lesion area covered by macrophages by nearly 50% (Table 2) (Figure IIA and IIB, available online at http://atvb.ahajournals.org). In contrast, probucol significantly increased the percentage lesion area that stained positive for collagen (Figure IIC to IIF) (66±13% versus 45±10% for probucol-treated versus control mice). At the descending aorta, probucol decreased total cell numbers, macrophages, and mRNA for F4/80 antigen by 90%, a value comparable to the extent of lesion inhibition (Table 2). Despite this, cells per lesion area remained unaltered, because lesions in the descending aorta of apoE–/– mice consisted almost entirely of macrophages (Table 2). Extracellular deposits of collagen were barely detectable, and probucol did not alter its content (Table 2).
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Discussion |
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The present study confirms6 that in apoE–/– mice probucol affects atherosclerosis in a site-specific manner, increasing lesion size at the sinus while at the same time almost completely preventing disease in the descending aorta. Here we used this unique feature as an experimental tool to address how probucol affects atherosclerosis. A novel finding of the present study is that neither the increased lesion size nor the disease-inhibiting effect of probucol can be explained readily by parallel changes in lipid oxidation, as assessed by the accumulation of CE-OOH, 7KC, or F2-isoprostanes at the different sites. Our results also show that whereas probucol increases the lesion size at the sinus, these lesions are more fibrous and contain, relative to lesion size, fewer inflammatory cells, ie, features that stabilize lesions and therefore can, overall, be seen as protective rather than disease-promoting.
Lipoprotein lipid oxidation in the vessel wall is commonly considered a cause of atherosclerosis, although recent observations suggest a dissociation between the 2 processes.5–9 The present study further supports such a dissociation. Most notably, it is the first study to our knowledge in which 3 separate measures of lipid oxidation were applied simultaneously and to both lesion size-enhancing and size-inhibiting conditions. Independent of the marker used, the extent of accumulation of lipid oxidation markers inversely correlated with lesion size, except for 7KC at the thoracic/abdominal aorta (Figure 3). Such inverse relationship is difficult to reconcile with the notion that lipid oxidation causes atherosclerosis, although we assessed lipid oxidation at only one time point during disease development, and as the accumulation of CE-OOH, 7KC, and F2-isoprostanes. However, previous studies in apoE–/– mice20 and humans26 indicate that with increasing disease severity there is net accumulation of aortic oxidized lipids (although oxidized lipids accumulate well after nonoxidized lipids). Thus, even if metabolism of oxidized lipids were to occur, formation of oxidized lipids appears to exceed such metabolism. Metabolism of oxidized lipids is also unlikely to explain our results, because in that case probucol would need to have increased metabolism at the aortic root and decreased it at the descending aorta, for which there is no evidence in the literature. Therefore, the simplest explanation of our data are that probucol’s effects are not explained by changes to aortic lipid oxidation, providing further evidence for dissociation between atherosclerosis and aortic lipid oxidation. Our data are also not immediately consistent with the central element of the oxidative modification theory of atherosclerosis,2 namely that LDL oxidation causes atherosclerosis, because our analyses also included CE-OOH, the single most abundant oxidized lipid in lesion lipoproteins including LDL,20,27 that is also formed in LDL exposed to 2-electron oxidants that favor apolipoprotein B-100 oxidation.28,29
From the inverse relationship between aortic lipid oxidation and lesion size, we do not conclude that lipid oxidation inhibits atherosclerosis. Rather, our contention is that the 2 processes are not causally linked. Previous studies have shown that in apoE–/– mice the aortic content of oxidized lipids, such as F2-isoprostanes30 and CE-OOH,20 increases with increasing lesion size and development. Our results are consistent with this. In control apoE–/– mice, the content of F2-isoprostanes and CE-OOH was higher at the sinus than thoracic/abdominal aorta (Table I and Table 1), and the rate of lesion development along the aortic tree differs, with sinus lesions being larger and more developed than those in the descending aorta.21 However, a direct relationship between lesion size and content of oxidized lipid does not prove a causal relationship between the two parameters.
Our studies show that probucol enhanced lesion size at the sinus by means other than increasing lipoprotein lipid accumulation. Similarly, we show that probucol decreased total cell and macrophage numbers per lesion area. By contrast, the drug substantially increased the lesion content of collagen. Given its preponderance, this increase in collagen can explain why probucol, overall, increased lesion size at the sinus. Our findings are consistent with previous reports on compositional changes caused by probucol in coronary arteries of hypercholesterolemic rabbits14,15 and nonhuman primates.13 In these animals, probucol caused more dense and fibrous plaques, with thicker caps and fewer macrophages. Importantly, these changes are associated with a more stable plaque type, less vulnerable to rupture, and in the rabbit have been reported to increase survival.15 Therefore, the action of probucol at the aortic sinus in apoE–/–, and possibly also LDL receptor–/–,31 mice may be considered beneficial rather than pro-atherogenic.
A striking feature is that in the descending aorta, probucol almost completely prevented atherosclerosis to an extent exceeding that reported for most other interventions. As discussed, this could not be explained by inhibition of lipid or lipoprotein lipid oxidation. Rather, the extent of disease inhibition was reflected in the decreased content of arterial macrophages and nonoxidized lipids. As probucol decreased macrophages (but not lipid) at the sinus and descending aorta, and lesions in the descending aorta are almost entirely composed of macrophages that contain essentially all lipoprotein-derived lipid, we speculate that this antiinflammatory action explains probucol’s antiatherosclerotic activity. Interestingly in this context, probucol monosuccinate, which prevents restenosis after percutaneous coronary intervention12 and is being tested as a novel therapeutic against atherosclerosis, acts as an antiinflammatory drug by reducing vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1.32 We are presently investigating whether the apparent antiinflammatory action of probucol is related to its ability to induce heme oxygenase-1.17,18,33
Unexpectedly, we observed the extent of probucol metabolism to be significantly increased in the aorta compared with the sinus (Figure 4), suggesting that some of the differences in action at the sinus versus descending aorta may be related to probucol metabolism. We do not know at present whether this relates to the previously reported antiinflammatory activity of probucol, ie, inhibition of lipopolysaccharide-induced secretion of interleukin-1 by macrophages.34 Little is known about probucol metabolism, although it was more extensive in the aorta than liver, as assessed by the respective ratio of bisphenol plus diphenoquinone-to-probucol (not shown), suggesting the involvement of processes other than those mediated by hepatic cytochrome P-450. Nonenzymatic oxidants can convert probucol to its bisphenol and diphenoquinone.24 We observed recently that during this process a bioactive intermediate is formed that protects vessels against oxidant-induced endothelial dysfunction,35 suggesting that probucol may act as a pro-drug. Preliminary results suggest that this probucol intermediate has antiinflammatory and antiatherosclerotic activities surpassing those of probucol (BW, PKW, KB, KC, Antony Lau and RS, 2005 unpublished).
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Acknowledgments |
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This work was supported by the Australian National Health & Medical Research Council (grants 970998, 151602, and 222722 to R.S.).
Received January 11, 2005; accepted June 3, 2005.
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