(Circulation. 1997;96:968-974.)
© 1997 American Heart Association, Inc.
Articles |
Both Dietary Fish-Oil Supplementation and Aspirin Fail to Inhibit Atherosclerosis in Long-term Vein Bypass Grafts in Moderately Hypercholesterolemic Nonhuman Primates
From the Department of Cardiothoracic Surgery, Medical College of Wisconsin, Milwaukee (L.E.B., G.N.O., G.H.A.), and the Department of Health Sciences, University of Wisconsin at Milwaukee (V.A.S.).
Correspondence to Lawrence E. Boerboom, PhD, Department of Cardiothoracic Surgery, Medical College of Wisconsin, 9200 W Wisconsin Ave, Milwaukee, WI 53226. E-mail lboerboo{at}post.its.mcw.edu
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Abstract |
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Background Aortocoronary vein bypass grafts are vulnerable to late atherosclerotic occlusion. Conventional platelet inhibitor therapy provides early but not persistent protection against graft failure. Evidence suggests that consumption of marine foods may reduce cardiovascular disease, possibly because of the unique long-chain unsaturated
-3 fatty acids
present in these foods. We hypothesized that dietary fish-oil
supplementation would protect against atherosclerosis
in vein bypass grafts. Methods and Results Thirty-three moderately hypercholesterolemic cynomolgus macaques were divided into four groups: control, control+aspirin, fish oil, and fish oil+aspirin. Each control group received olive oil as placebo to equalize calorie and fat consumption with that of the fish-oil groups. Both oils were in ethyl ester form, with the fish oil providing 0.88 g/d eicosapentaenoic acid. The aspirin dose was 40 mg/d. Cephalic vein grafts were interposed bilaterally in the carotid arteries and excised for analysis at 4 years. Bleeding time was significantly prolonged in all groups receiving fish oil or aspirin (P<.05). Plasma cholesterol levels were similar among groups, averaging 6.9±2.4 mmol/L (267±94 mg/dL). The extent of atherosclerosis in vein grafts did not differ among groups as evaluated both by Sudan IV staining of intimal lipid lesions (27±21% of total surface area, P=.89) and analysis of cholesterol content (236±203 nmol/mg, 9.1±7.8 µg/mg, P=.85). Vein graft connective tissue composition was also unaffected by treatment.
Conclusions Our findings do not support the use of concentrated dietary fish-oil supplements or aspirin for the prevention of atherosclerosis in long-term vein bypass grafts. Consumption of fish flesh or less refined oil preparations could have effects different from those of the purified fish-oil ethyl esters we used.
Key Words: atherosclerosis • bypass • coronary disease • fish oil • grafting • aspirin
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Introduction |
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Atherosclerotic degeneration is the major cause of late aortocoronary vein graft failure.1 Early graft patency rate was improved in humans by platelet inhibition with aspirin and dipyridamole.2 We have shown in a nonhuman primate model that platelet inhibition also reduces early graft lipid content.3 However, it appears that whereas platelet inhibition may slow the rate of progression of graft atherosclerosis, it does not prevent it, such that the graft cholesterol level is similar in our model at 18 months in treated and untreated monkeys.4 This underscores the need for an alternative therapy.
Evidence derived from epidemiological studies of Eskimos who consume
large quantities of marine mammals,5 6 7 clinical
observations,8 9 and experimental animal
studies10 11 have suggested that consumption of a
marine-rich diet or dietary supplementation with fish oil may reduce
cardiovascular disease. These benefits have been
attributed to the long-chain highly unsaturated -3 fatty acids found
in fish, eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA). Potential mechanisms postulated to be
responsible for the effects of fish oil include alterations in
platelet function,12 lipid
metabolism,13 and eicosanoid and
leukotriene metabolism.14
Nonetheless, beneficial findings have not been demonstrated in all
studies of the relationship between fish oil and
cardiovascular disease.15 16 17 18
The influence of -3 fatty acids on vein bypass graft lipids has not
been investigated in a primate model. The present study in
cynomolgus monkeys was designed to determine whether dietary fish-oil
supplementation, alone or synergistically with aspirin, reduced
atherosclerosis in long-term vein grafts.
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Methods |
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Animal Model, Diet, and Treatment Regimen
Thirty-three adult male cynomolgus macaques (Macaca fascicularis) (8.1±1.1 kg, mean±SD) were fed a moderately atherogenic diet (Purina 5045-3) designed to produce plasma cholesterol levels in the high-normal range for humans. Fish meal was excluded from this chow to avoid extraneous sources of dietary
-3 fatty acids. The diet contains 12% fat and 0.18% added
cholesterol by weight and provides 3.7 kcal/g, with 26% of
calories derived from animal fat (tallow).
Monkeys were divided randomly into four treatment groups: control, n=9;
control+aspirin, n=8; fish oil, n=9; and fish oil+aspirin, n=7. Each of
the control groups received olive oil as a placebo to control for the
additional caloric and fat intake that fish oil contributed to total
dietary consumption. Aspirin-treated animals received 40 mg/d. Fish oil
and olive oil were in the ethyl-ester form and were provided in gel
capsules by the Biomedical Test Material Program of the National
Institutes of Health (produced by the National Marine Fisheries
Service, Charleston, SC). Antioxidant levels were balanced in the two
oil preparations. Each animal received two 1-g capsules of the
respective oil per day. The fish-oil treatment provided 0.88 g/d EPA
(1.36 g/d EPA+DHA, 1.57 g/d total -3). This dose was selected to be
equivalent on a body-weight basis to the consumption of EPA by
Greenland Eskimos.19 20 All medications were given orally,
and compliance was ensured by observing the monkeys and withholding
food until the medications were consumed. The atherogenic diet was
started 3 months before vein bypass grafts were inserted, oil
supplementation 2 months before insertion, and aspirin 2 weeks before
insertion.
Surgical Procedure, Blood Collection, and Bleeding Time
Animal preparation and surgery were performed in a manner
similar to that previously described,21 with the
exceptions that the cephalic vein segment used for grafting was
distended at 150 mm Hg for 1 minute, anticoagulation was
performed with heparin 1500 U, grafts were placed bilaterally in the
carotid arteries, and the segment of artery between anastomoses was
excised after ligation. The protocol regarding animal
maintenance, handling, and surgical operations followed in this
study was approved by the Animal Care and Use Committee of the Medical
College of Wisconsin.
Blood was collected for analysis before the beginning of the atherogenic diet and at 1, 2, and 4 years.
Bleeding time was evaluated on the medial aspect of the forearm at the termination of the study (Simplate-II bleeding time device, General Diagnostics). Duplicate determinations were made on separate days and averaged.
Graft Removal and Processing
Grafts were excised for analysis 4 years after
insertion. Segments of ungrafted cephalic vein were also excised for
analysis at that time. The vessels were gently flushed with
PBS, and the right graft was fixed at 100 mm Hg intraluminal
pressure with modified Karnovsky's fixative.22 The
anastomotic sites were removed from the left graft and excluded from
the chemical analysis. The left graft and cephalic vein segment
were blotted dry, weighed, and frozen at -70°C for later processing
and chemical analysis. At the time of processing, the graft was
thawed and homogenized on ice with
homogenizing buffer23 in a glass
homogenizer operated manually to avoid heat
production.
Sudan IV Staining, Photography, and Morphometry
After fixation, the right carotid grafts were stained for lipid
with Sudan IV as described previously24 to demarcate lipid
lesions at the luminal surface. All grafts were stained
simultaneously in the same solution to ensure uniform
staining conditions among specimens. The grafts were slit open
longitudinally after staining, pinned luminal side up to a cork board,
covered with cacodylate buffer solution to avoid desiccation, and
photographed. Photographs were printed on 8x10-in paper. The total
surface area of the graft and the area of the intimal surface that had
lipid lesions were measured by separately tracing the perimeter of the
graft and the lesion areas with a digital planimeter. Graft width was
determined at five or more uniform intervals, and the values were
averaged to obtain mean luminal circumference for each specimen.
Chemical Assays
All assays were performed in duplicate, and the results were
averaged; if duplicates varied by >10%, the assay was repeated.
Enzymatic kits were used to measure total and nonesterified
cholesterol (Cholesterol CII and Free
Cholesterol C, respectively; Wako Chemicals) and
triglycerides (334-UV, Sigma Chemical Co).
Phospholipid,25 26 hydroxyproline,27 and
glycosaminoglycans (with chondroitin sulfate A as
standard, Sigma)28 were measured nonenzymatically
according to previously described methods. Graft data are expressed as
units/mg wet wt. "Collagen" and "elastin" values are
expressed as units of hydroxyproline. Assays of plasma and grafts were
performed by the same procedures. Plasma fatty acids were
transesterified by slight modification of a standard sulfuric
acid–catalyzed method,29 and fatty acid methyl esters
were quantified by gas chromatography.
Statistical Analysis
Data were evaluated by ANOVA, and if a significant difference
was observed among groups, either the Student-Newman-Keuls test or
Dunn's method was used to evaluate for differences between specific
groups, depending on whether the data were normally distributed. Values
of P<.05 were considered to be statistically
significant.
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Results |
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The atherogenic diet and oil medications modified plasma lipids. Total plasma cholesterol was 2.4±0.6 mmol/L (92±22 mg/dL, mean±SD) before the beginning of the diet and increased to 6.9±2.4 mmol/L (267±94 mg/dL) with diet consumption. Plasma lipid values observed during the diet are shown in Table 1
as the average of measurements made at 1, 2, and 4
years. There were no statistically significant differences among groups
in any of these variables at any single time interval or among
intervals. If the data are analyzed as the two fish-oil groups
pooled together versus the pooled control groups, both LDL
cholesterol and the LDL/HDL ratio became significantly
greater in the groups treated with fish oil (both P<.05).
HDL cholesterol difference among groups does not achieve
statistical significance in such an analysis. The influence of
the oil treatments on plasma fatty acids is shown in Table 2. There was significantly more EPA and DHA in the two
groups receiving dietary fish-oil supplementation than in the control
groups (P<.05). This augmentation of the -3 fatty acids
occurred in conjunction with a displacement of
arachidonic acid (P<.05).
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Overall platelet hemostatic function was significantly altered in
the groups receiving fish oil with or without aspirin and in the
control+aspirin group compared with the control group without aspirin,
as demonstrated by prolonged bleeding time (Fig 1, P<.05). Bleeding time did not differ between the fish-oil
and fish-oil+aspirin groups.
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All grafts were patent at the termination of the study. Staining with
Sudan IV revealed that lipid lesions covered similar amounts of the
intimal surface in all groups: 24±17% in the control group, 31±32%
in the control+aspirin group, 29±17% in the fish-oil group, and
24±19% in the fish-oil+aspirin group (Fig 2, P=.89). There was no significant treatment
effect on graft luminal circumference, which was 11.0±2.0 mm in
the control group, 9.4±2.7 mm in the control+aspirin group,
10.3±2.9 mm in the fish-oil group, and 9.2±3.4 mm in the
fish-oil+aspirin group (P=.52).
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The changes in total cholesterol concentration that
occurred in cephalic veins during the course of the study, both as
normal ungrafted veins and as grafts, are illustrated in Fig 3. Vessel cholesterol levels were 24±5
nmol/mg (0.9±0.2 µg/mg) in veins at the time of harvesting for
insertion as grafts. In ungrafted veins, this increased slightly but
significantly by the end of the study, to 36±9 nmol/mg (1.2±1.0
µg/mg) (P<.05). A much greater increase, to 236±203
nmol/mg (9.1±7.8 µg/mg) (average of all groups), occurred in vein
grafts (P<.0001). Graft lipid values for individual groups
are shown in Table 3. Cholesterol and
phospholipid levels were similar among treatment groups.
Triglyceride was significantly greater in both
aspirin-treated groups than in the groups not receiving aspirin
(P<.05). The portion of total cholesterol that
was esterified was greater in grafts, 31±16%, than in ungrafted
veins, 21±12% (P<.01). The esterified
cholesterol fraction in grafts did not differ among groups
(P=.75).
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The range among the animals in plasma cholesterol
response to the atherogenic diet was 3.9 to 14.0 nmol/L (149 to 539
mg/dL), with a coefficient of variation of 35%. Because of the
well-documented relationship between plasma cholesterol and
the extent of atherosclerotic disease, we therefore performed linear
regression analysis of the cholesterol levels in
plasma versus grafts (Fig 4). This demonstrated a
significant, direct relationship between these two variables
(P<.0001, r=.70). Regression analysis
demonstrated that graft cholesterol was also directly
related to the ratio of plasma total/HDL cholesterol
(P<.01, r=.56). However, neither LDL
(P=.31, r=.21) nor HDL (P=.19,
r=.27) alone achieved a significant relationship with graft
cholesterol. ANCOVA showed that the relationship between
graft and plasma cholesterol was similar in all groups.
There was a close relationship between graft cholesterol
and the percentage of the intimal surface that stained for lipid
lesions with Sudan IV (P<.001, r=.70).
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Data for graft collagen, elastin, and
glycosaminoglycans are shown in Table 3
. None of
the treatments produced a significant difference among these connective
tissue components of the extracellular matrix.
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Discussion |
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There is no experimental animal model that precisely mimics the human condition of atherosclerotic disease in every detail, but certain nonhuman primates develop atherosclerotic lesions that bear a remarkable resemblance to those seen in human beings. This appears to be due in part to the similarity in lipid and lipoprotein metabolism and vascular biology between monkeys and humans that is not shared by most other species. We selected the cynomolgus macaque for this study because it is regarded as one of the best models for studying human atherosclerosis. In this species, lesions occur naturally and are morphologically similar to those of human beings; they are exacerbated by dietary cholesterol; their males, like humans, are more susceptible to coronary artery lesions than are females; they have the greatest incidence of myocardial infarction among the macaques; and their lipoprotein distribution is similar to that of humans.30
We found no evidence in this study that either dietary fish-oil supplementation or aspirin, alone or in combination, reduced atherosclerosis or improved vessel caliber in long-term vein bypass grafts as assessed by chemical analysis, by the fraction of the intimal surface containing lipid lesions, or by graft luminal circumference. This confirms our concern that platelet inhibition with conventional therapy may be of diminishing benefit late after operation.4 It also comes amid a mounting number of recent reports15 18 31 32 suggesting that fish oil has failed to fulfill early enthusiastic expectations that it might prevent cardiovascular disease.
Fish oil did not modify total plasma cholesterol levels in our monkeys and did not change LDL or HDL cholesterol significantly, although there was a trend for LDL to be greater and HDL to be lower than in control animals. An increase in LDL with fish-oil supplementation has been reported.33 Whether the magnitude of this change is significant or merely a trend (as in our study) may be a dose-related phenomenon. It is noteworthy that decreased HDL cholesterol levels have been reported in previous studies of fish oil carried out in nonhuman primates.11 34 This observation is cause for concern because of the known inverse relation between HDL and coronary artery disease. Within each group, there was a range in the plasma cholesterol response to the atherogenic diet. We showed that graft cholesterol was directly related to the plasma cholesterol levels and that plasma cholesterol accounted for the vast majority of the variation in graft cholesterol. This relationship is in agreement with the relation between plasma cholesterol and both the risk of graft occlusion1 and heart disease in humans.35 There was no significant difference in this relationship among treatment groups as evaluated by ANCOVA.
Bleeding time provides a good assessment of overall platelet function. We observed a prolongation of bleeding time with either fish oil or aspirin compared with the control group without aspirin. This confirms that our monkeys consumed their medication, that it was absorbed, and that the dose was adequate to alter platelet function. Treatment effects may also have occurred in other parameters of platelet function and in biological activity of other cell lines that were not monitored.
Although quantitative evaluation of graft lipids as influenced by fish oil has not been reported previously, cod-liver oil or Max EPA has been reported to decrease intimal thickening in vein bypass grafts.36 37 38 On the other hand, cod-liver oil, aspirin, and dipyridamole all failed to reduce intimal hyperplasia in venoarterial allografts when these agents were given either individually or combined.17 All of these studies were performed in dogs, a model in which induction of atherosclerosis is virtually impossible. It should also be emphasized that both intimal proliferation and lipid deposition are commonly observed in atherosclerosis but are not necessarily linked, particularly in vein bypass grafts. Neither our lipid data nor our connective tissue data evaluate exactly the same phenomenon that was evaluated in measurements of intimal thickening in the above-mentioned studies. In animals, such as monkeys, that develop atherosclerosis, intimal lesions are composed of cellular, lipid, and connective-tissue components. Our data demonstrate that neither lipid nor connective tissue was reduced by fish oil or aspirin in our grafts. Taken together, these findings appear to be in contrast with the studies in dogs cited above. Apart from the difference in species, other possible explanations for this contrast include differences in the type of fish oil used, dose, and the much shorter duration of the canine studies.
The influence of fish oil on arterial
atherosclerosis has been evaluated in a variety of
studies. Weiner et al10 showed in
hypercholesterolemic pigs that cod-liver oil decreases
lesion size in both abraded and nonabraded coronary arteries.
However, a study of rather similar design but using -3 ethyl esters,
like our study, failed to find a benefit.39 In rhesus
monkeys fed a diet containing menhaden oil and coconut oil in various
proportions, atherosclerosis was reported to be
decreased by greater amounts as the proportion of menhaden oil
increased relative to coconut oil.11 But since coconut oil
is known to be highly atherogenic, it is not clear whether the change
was derived from increasing the proportion of fish oil or lowering the
proportion of the atherogenic coconut oil. Also in the later study,
serum cholesterol in the group receiving only coconut oil
was approximately double that of the groups receiving fish oil. Fish
oil was also found to decrease the percentage of the aortic but not the
carotid luminal surface containing lesions in African green monkeys
that were fed diets in which the fat source was either lard or fish
oil.34 Total plasma cholesterol and HDL
cholesterol were both significantly lower in their fish-oil
group. Although two different diets with different fat sources were
used in the later study, the authors report that the portions of
saturated, monounsaturated, and polyunsaturated
fatty acids were equal in the lard and fish-oil groups. In our study,
all animals consumed the same diet and only the oil treatments, which
were provided as a dietary supplement, were varied. They also report a
daily dose of 3.8 g total -3 fatty acids, compared with
1.57 g in our study (0.69 versus 0.19 g/kg). The high dose they
achieved was presumably possible because the fish oil was contained in
the diet and served as the dietary fat source. The animals were thus in
essence forced to accept this dose.
Limitations of our study include the possibility that both olive oil
and fish oil might have had effects of equal magnitude and direction
and that therefore no difference between treatments was detected.
However, omission of an oil control would have raised the problem of
calorie and fat consumption being unequal among groups. Another
limitation is that only one dose was studied. As discussed in
"Methods," we specifically selected our dose to match the -3
fatty acid consumption of Eskimos. This dose is as great as or greater
than in studies that reported a positive effect from fish oil, but it
cannot be ruled out that in our model, a more pharmacological dose
might have produced a different outcome. We demonstrate that our dose
altered plasma fatty acids, but we did not monitor cellular
incorporation of -3 fatty acids. Interestingly, it has been
suggested that the dose-response relationship could fit a U-shaped
model.40 Finally, since the number of animals was limited
to 7 to 9 per group, there is the possibility that treatment effects
were too subtle to be detected by the statistical power inherent in
this study. However, because the data demonstrate that the influence of
plasma cholesterol is much stronger than the influence of
which oil was consumed, it seems very doubtful that increasing sample
size within practical limits would have revealed a treatment
effect.
In previous studies, we observed that aspirin and dipyridamole reduce the rate of early lipid deposition in vein grafts.3 However, by 18 months it was beginning to become apparent that this early benefit may be obliterated with time, as the lipid level in treated grafts catches up with that in untreated grafts.4 The present study confirms and extends these findings, demonstrating that at 4 years there is no difference in graft cholesterol between the control groups with and without aspirin. This lack of benefit occurs despite a persistent effect on platelet function. Curiously, graft triglyceride levels were greater in our aspirin-treated animals. It is not clear what accounts for this observation, but it appears to be unrelated to plasma triglyceride levels, which were similar in all groups.
Our findings do not support the use of concentrated dietary fish-oil
supplements for the prevention of atherosclerosis in
vein bypass grafts. Caution must be taken to emphasize that consumption
of fish flesh or less refined oil preparations could have effects
different from those of the purified fish-oil ethyl esters we used. The
report of the Third Conference on Antithrombotic Therapy by the
American College of Chest Physicians states that there is no evidence
to support the use of concentrated -3 fatty acids in fish oil for
the prevention or treatment of coronary artery
disease.41 Still, it is possible that there is some
uniquely beneficial nutrient in fish that is not as yet identified and
that may not be present in highly purified fish oils. The possible
benefit in eating fish and the validity of the early Eskimo
epidemiological studies are supported by the more recent observation
that as the Eskimo lifestyle and diet have become more Westernized,
their lipoprotein pattern has changed to a less favorable
profile.42 Since benefits have been reported from eating
fish in such small quantities that only very modest amounts of -3
fatty acids would be provided,7 one is left with the
suspicion that other constituents in fish may contribute to their
effects. Indeed, the Nutrition Committee of the American Heart
Association recommends the regular consumption of fish but found no
justification for the ingestion of fish-oil
capsules.43
The impressive development of classic degenerative lesions in these primate vein grafts and their direct relation to acquired plasma cholesterol speaks strongly for risk factor intervention specifically to reduce total cholesterol and to increase HDL as first-line preventive therapy against late graft atherosclerosis.44 Subtler perturbations may simply not have the power to overcome the strong influence of plasma cholesterol on vein graft atherosclerotic changes.
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Acknowledgments |
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This work was supported by grant HL-41840 from the National Institutes of Health to Dr Boerboom. The technical assistance of Diane Munzenmaier, Barbara Truitt, Connie O'Connor, and Gary Fehl, PA, is gratefully acknowledged. We also greatly appreciate the secretarial assistance provided by Mary Lynne Koenig.
Received September 23, 1996; revision received February 3, 1997; accepted February 7, 1997.
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