(Circulation. 1997;96:3593-3601.)
© 1997 American Heart Association, Inc.
Articles |
Vitamins C and E Inhibit O2- Production in the Pig Coronary Artery
From the Department of Medicine (Cardiology Division), University of Washington, Seattle (B.C.B.), and the Departments of Medicine (Andreas Gruentzig Cardiovascular Center) (G.L.N., K.R., A.K., S.B.K.) and Pathology (D.S.S.), Emory University School of Medicine, Atlanta, Ga. Dr Nunes is now at Dante Pazzanese Cardiology Institute, São Paolo, Brazil.
Correspondence to Bradford C. Berk, MD, PhD, Division of Cardiology, 357710, University of Washington, Seattle, WA 98195. E-mail bcberk{at}u.washington.edu
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Abstract |
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Background We previously found in a pig coronary balloon injury model that vitamins C and E as well as probucol had beneficial effects on the vessel response to injury measured by morphometry. These effects correlated with an inhibition in the ability to oxidize LDLs ex vivo, suggesting that the morphological response was due to the antioxidant effect of the treatments.
Methods and Results In the present study, the
production of O2- by vessels 14 days
after balloon injury was determined and correlated with circulating and
tissue levels of vitamins C and E. Twenty-five domestic pigs were
divided into four groups: control (n=7), vitamin C (500 mg/d, group C,
n=6), vitamin E (1000 IU/d, group E, n=6), and vitamins C and E (500
mg/d and 1000 IU/d, group C+E, n=6). Vitamins were administered 7 days
before oversized balloon injury of the left anterior descending
coronary artery (LAD) and continued for 14 days after injury.
Vitamin C and E concentrations were determined in plasma and
lymphocytes as an index for tissue levels. Vessels were harvested after
animals were killed, and O2-
production was measured by lucigenin chemiluminescence.
O2- production by the injured LAD was
2.5-fold greater than O2- production
by the uninjured LAD or right coronary artery (RCA). The
increase in O2- was caused primarily by cells
present in the media and neointima. All vitamin-treated
groups showed significantly decreased O2-
production in both the RCA and LAD (
45% inhibition)
relative to vessels in the control, untreated group. There was a
significant correlation between LAD O2-
production and lymphocyte vitamin E levels.
Conclusions The present study is the first to show increased O2- production in injured vessels and to demonstrate that antioxidant vitamins reduce O2- production. These results suggest that beneficial effects of antioxidant vitamins in coronary artery disease are related, in part, to alterations in vessel redox state.
Key Words: antioxidants • restenosis • free radicals • vasculature
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Introduction |
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An evolving concept in the pathogenesis of cardiovascular diseases such as atherosclerosis, hypertension, and restenosis after angioplasty is a causative role for oxidative stress mediated by increased levels of reactive oxygen species (H2O2, O2-, and OH-).1 Numerous animal and human studies show that administration of antioxidants limits the extent or clinical severity of atherosclerosis. For example, both probucol and BHT limit atherosclerosis and VSMC growth in hyperlipidemic models of atherosclerosis.1 2 3 Human epidemiological studies suggest that high dietary intake of natural antioxidants such as vitamins C and E reduces the risk of cardiovascular mortality.4 5 It has been inferred from these studies that the beneficial effects of the antioxidants (vitamins C and E, probucol, and BHT) were related to their ability to decrease production of O2-, but no measurements of vascular redox state were performed to prove this mechanism.
Oxidative stress also appears to be an important component of the
vessel response to injury, and the vessel redox state may contribute to
restenosis after coronary interventions. Reactive
oxygen species are present in large quantities during and
immediately after arterial injury.1 6 7
Although there have been many studies of the effect of probucol on the
vessel response to injury,8 9 probucol is not a
"pure" antioxidant and has important effects on interleukin
biology and cholesterol levels. In contrast, vitamins C and
E are more specific in their effects as antioxidants. Three groups,
including our own, have shown that antioxidant vitamins alter the
vessel response to injury in animals. We found that vitamins C and E
promoted vessel "remodeling" in the pig coronary artery,
as shown by an increase in vessel and lumen diameter.10
Konneh et al11 showed that in rats receiving a 0.5%
vitamin E plus 1% cholesterol diet, neointimal
thickening after carotid injury was reduced by 30% compared with
animals receiving either cholesterol alone or a control
chow diet. LaFont et al12 showed that in
hypercholesterolemic rabbits receiving vitamin E, there
was a significant improvement in the response to balloon injury of
femoral arteries, with 50% reductions in intima-media area and in
the loss of lumen area compared with untreated animals. Few studies
have analyzed the effects of vitamins C and E on the response
of human coronary arteries to balloon angioplasty. A small
study of vitamin E after angioplasty showed a benefit as measured by
exercise thallium testing 6 months after the procedure.13
Tardif et al,14 in a preliminary report from the MVP
Study, compared the effects of probucol and multivitamins
(ß-carotene, vitamin E, and vitamin C) alone and in combination in
patients undergoing angioplasty. They found a significant decrease in
restenosis with probucol but not with the
multivitamins.14 These studies, in combination, suggest
that antioxidant vitamins modify the vascular response to injury, which
we propose is related to decreased O2-
production.
Two potential mechanisms for beneficial effects of antioxidant vitamins on vessel function are improved endothelial cell function measured by increased vasodilation in response to endothelium-dependent vasodilators15 16 and decreased VSMC production of O2-. We propose that these effects on endothelial and VSMC function will be manifested by greater outward remodeling,17 defined by increases in vessel lumen and area without changes in media and intima mass. The importance of these mechanisms is suggested by the findings that states of hypercholesterolemia are associated with both increased production of reactive oxygen species and endothelial dysfunction.16 Lowering circulating LDL pharmacologically in hypercholesterolemic states improves endothelial function, as measured by vasorelaxation in response to acetylcholine.15 18 An additional improvement in vasorelaxation is observed when antioxidants are administered in addition to lipid-lowering therapy.15 The finding that nitric oxide, a critical regulator of vascular function, is inactivated by reactive oxygen species, which are present in higher levels in hypercholesterolemia, suggests that the balance between nitric oxide and reactive oxygen species may modulate the pathogenesis of atherosclerosis and the vessel response to injury. Recently, an important role for vascular remodeling has been proposed in the pathogenesis of atherosclerosis and hypertension and the response to vascular injury.17 19 20 Previous studies in animals21 have suggested a critical role for endothelial cells in remodeling, implying that the beneficial effects of antioxidants on the vascular response to injury are due to improved endothelial function.
These previous studies suggest that in hypercholesterolemia, as well as after vascular injury, an oxidizing environment must exist in the vessel wall that may influence vascular remodeling. In fact, the induction of NADH oxidase by angiotensin II22 and the importance of reactive oxygen species production in signal transduction by growth factors such as platelet-derived growth factor23 suggest that there may be long-term alterations in the capacity of the vessel to produce reactive oxygen species. Thus, the major hypotheses of the present study are that (1) in response to vascular injury, there will be a sustained increase in production of reactive oxygen species and (2) administration of antioxidant vitamins, which we have previously shown to promote outward vessel remodeling,10 should be associated with a significant reduction in vessel production of reactive oxygen species. To prove these hypotheses, we used the well-characterized pig coronary artery balloon injury model.9 10 We found a 2.5-fold increase in O2- production 14 days after injury of the LAD. The major source of O2- production appeared to be VSMCs. Furthermore, we showed that the increase in O2- was significantly inhibited by administration of vitamin C, vitamin E, and the combination of vitamins C and E, with no significant differences between treatments. The level of O2- production was associated with plasma and lymphocyte concentrations of vitamins C and E. These results suggest that the effects of vitamins C and E on production of O2- in the injured vessel are related to their antioxidant properties.
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Methods |
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Animals
Twenty-five juvenile female domestic swine were used in this study (weight between 20 and 34 kg; mean, 26.4±3.9 kg). The laboratory values for lipids and vitamin levels (Table
) are similar to those we reported
previously.10 Pigs were treated starting 1 week before
coronary balloon injury (day -7) with vitamin C 500
mg/d (group C, n=6), vitamin E 1000 IU/d (group E, n=6), both
(group C+E, n=6), or neither (control, n=7). These levels of vitamins
were chosen on the basis of two factors. First, at these doses there
should be saturation of endogenous stores and attainment of
a steady-state concentration based on previous studies.24
Second, these doses approximate those used in many human
studies.13 14 Vitamins were given daily mixed with the
chow and corn syrup, and the animals were observed to ensure that the
vitamins were taken. A normolipemic diet (containing 2.2 ppm carotene,
54 IU/kg vitamin E, and no vitamin C) was given to all animals. All
experimentation and animal care conformed to the National Institutes of
Health and American Heart Association guidelines for the care and use
of animals. The study was approved by the Emory University Animal Care
and Use Committee.
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Experimental Protocol
After 7 days of vitamin supplements (day 0), the animals
underwent coronary overstretch balloon injury exactly as
previously described.9 10 We chose the overstretch balloon
injury model for several reasons. The model has been extensively
characterized in our laboratories9 10 25 and provides a
very uniform increase in neointima mass with complete
endothelial regeneration. In addition, we previously
showed that vitamins C and E were associated with outward remodeling in
this model,10 suggesting that the pig coronary
artery is responsive to antioxidants. Coronary injury was
achieved by deliberate stretch of the target vessel with a
3.5-mm-diameter, 20-mm-long polyethylene terephthalate balloon catheter
(USCI). Three 30-second inflations at 10 atm were performed in the
proximal portion of the LAD. Inflations were separated by a 1-minute
interval to allow for coronary perfusion. After repeat
coronary angiography, the catheters were removed and the
cutdown was repaired. Nitroglycerin ointment (1 inch)
was applied topically, and the animals were allowed to recover.
Vessels were harvested 21 days after the initiation of treatment (day +14). We have previously shown that there is complete endothelial regeneration 14 days after injury and near cessation of VSMC proliferation.25 In addition, changes in vessel structure are nearly complete at this time, resulting in less variability in morphological measurements. Although there may be more leukocyte invasion at early time points, we believe that chronic increases in reactive oxygen species may be more important, as shown by Rajagopalan et al26 for hypertension. To harvest vessels, the animals were injected with heparin (200 U/kg IV) and killed by a lethal dose of pentobarbital (65 mg/kg). The heart was then rapidly excised through a left thoracotomy, and the coronary system was perfused with normal saline. The injured segments of the LAD (6 in groups C, E, and C+E and 7 in control) and uninjured RCA (same numbers as for LAD) were then carefully dissected from the epicardium, placed in a solution of modified Krebs/HEPES buffer, and further cleaned of all fat and adventitia under a dissecting microscope for assay of O2- production. Histochemical analysis of cleaned vessels showed that >90% of the adventitia (measured by presence of nerve and adipose tissue) was absent. Removal of adventitia was essential, because trapped blood cells (especially leukocytes and red blood cells) caused large increases in O2- production.
Vitamin and Lipid Measurements
Blood samples for determination of vitamin levels, plasma
cholesterol, and triglycerides were collected
at day -7 (before vitamin supplements), day 0 (day of balloon injury),
and day +14 (vessel harvest). Blood was collected in tubes containing 1
mg/mL EDTA, and for vitamin E assay, 4.4 µg/mL BHT was
added. Plasma was prepared by low-speed centrifugation
at 4°C. For vitamin C assay, 0.5 mL plasma was added to 2 mL
metaphosphoric acid (0.06 g/mL). Vitamin C and E levels were
determined as described previously on all animals.10
Lymphocytes were prepared from one half of the animals by Ficoll
Hypaque and, after washing, were frozen for transport to the University
of Washington. Vitamin C and E levels were measured by
microspectrophotometric methods.27 28
Lucigenin Assay of Vessel O2-
Production
Vessels were equilibrated for 1 hour at 25°C in Krebs-HEPES
buffer (in mmol/L: NaCl 99.01, KCl 4.69, CaCl2
1.87, MgSO4 1.2, K2HPO4 1.03,
NaHCO3 25.0, Na-HEPES 20.0, and glucose 11.1, initially
gassed with 95% O2/5% CO2, pH 7.4). For
measurement of O2-, the vessels were
transferred to a glass scintillation vial containing 2 mL fresh
Krebs-HEPES buffer with 0.25 mmol/L lucigenin
(bis-N-methylacridinium nitrate). Chemiluminescence was
measured with a scintillation counter (LS 7000, Beckman Instruments) in
out-of-coincidence mode with a single active photomultiplier tube.
Counts were performed for 1 minute every 2 minutes until a plateau was
reached (usually 15 or 16 minutes). Standardization between different
experiments and conversion into moles of O2-
was performed with xanthine/xanthine oxidase as
described.16 All values were expressed relative to tissue
dry weight (48 hours at 48°C). The lucigenin assay is very
reproducible, and production of O2- is
quite stable, as shown by the fact that remeasurement of
O2- in the same vessels 1 hour later yielded
values that differed from the initial values by <5% (n=4). The major
limitations of the lucigenin assay are that it does not readily
separate intracellular and extracellular O2-
production, nor can it determine the enzymatic source of
O2- production. The relative
contributions of extracellular and intracellular production of
O2- were determined by comparison of the
inhibitory effects of exogenously added superoxide
dismutase (1000 U/mL) and Tiron (10 mmol/L
4,5-dihydroxy-1,3-benzene disulfonic acid). Tiron completely inhibited
lucigenin chemiluminescence, whereas superoxide dismutase caused an
65% decrease in chemiluminescence. Thus, the majority of
O2- is produced by intracellular
mechanisms.
Statistical Analyses
All experiments were performed in a blinded fashion. Data are
reported as the mean±SEM. We compared data by a one-way ANOVA (vessel
and treatment). To compare group means after computation of the ANOVA,
a contrast was set up to yield F and P values. Post hoc
analysis was performed with the Tukey-Kramer Honestly
Significantly Different test of pairwise mean comparisons. For
analysis of correlations between vitamin concentrations and
O2- production, the logs of
O2- values were compared with concentration,
and Spearman correlation coefficients were determined with Bonferroni
correction. The statistics were computed with the program
SYSTAT. A value of P<.05 was considered
significant.
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Results |
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Group Characteristics and Hematologic Values
Characteristics of the swine used for the present study are listed in the Table
. There were no significant differences in animal
weight among the groups at the time of balloon injury. No significant
differences in white blood cell or lymphocyte numbers were observed
between days -7 and +14 or among the treatment groups (Table).
Plasma Cholesterol and Vitamin Levels
Total plasma cholesterol and triglycerides
were unchanged throughout the study period in all groups (Table 1), as
previously reported.10 Care was taken to store the plasma
and white blood cells at -80°C rapidly in the presence of
metaphosphoric acid to stabilize vitamin C. Plasma vitamin C levels
(Table 1) increased in both groups C and C+E at day +14. The increase
was significantly greater in group C (2-fold increase). Animals that
received vitamin E supplementation (groups E and C+E) showed an
2.5-fold increase in plasma vitamin E levels. Lymphocytes and
neutrophils accumulate vitamin C intracellularly29 30 and
may more accurately reflect tissue levels of vitamin C. There was a
significant increase in lymphocyte vitamin C levels in group C, which
correlated with the increase in plasma vitamin C. However, there was no
significant increase in lymphocyte vitamin C in group C+E. There were
significant increases (4-fold) in lymphocyte vitamin E levels in
both groups E and C+E. This increase was greater than that observed in
plasma vitamin E levels (2-fold), reflecting the partitioning of
vitamin E into the lipid-rich cellular environment.
Vessel Production of O2-
Vessel redox state was assessed by measurement of vessel
O2- production with a lucigenin
chemiluminescence assay. The value for O2-
production at 16 minutes corrected for vessel dry weight was
used for analysis as previously described.16
Initial studies showed that there was no significant difference in the
production of O2- by the uninjured LAD
compared with the uninjured RCA (Fig 1, P>.1). Thus, for all
subsequent experiments, the uninjured RCA from each animal served as a
control for the injured LAD to document the effect of injury and to
normalize for the effect of treatment.
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The production of O2- by the injured
LAD segment compared with the uninjured LAD segment was then
analyzed. The entire LAD was carefully removed, and by visual
inspection, a 1-cm portion from the injured area was removed. Uninjured
1-cm segments proximal and distal to the injured LAD were also
prepared. The segment of vessel designated as having been injured was
the predominant source of O2-
production, with a 3-fold increase compared with the uninjured
proximal or distal segments (Fig 2, P<.001). This result
indicates that O2- production is
significantly increased 14 days after balloon injury. In addition,
there was no effect of injury on O2-
production distal to the site of balloon inflation, unlike the
effect of injury in the rat carotid artery on cell
proliferation.31
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The source of O2- was then determined in
control animals that received no vitamins. The entire vessel was
carefully removed to preserve endothelium, and
O2- production was measured. The
vessel was then reequilibrated in Krebs-HEPES buffer for 30 minutes,
the endothelium was gently removed, and
O2- production was measured again.
Control experiments showed that there was no significant change in
O2- production during the 30-minute
period of reequilibration of vessels (with or without
endothelium, not shown). There was very little
difference in O2- when the
endothelium was removed (Fig 3). For example, after removal of
endothelium, O2-
production decreased by 10% (eg, from 4437±910 to
3993±677 cpm/µg in the uninjured LAD and from 9708±2017 to
9002±1846 cpm/µg in the injured LAD at 15 minutes of incubation).
These findings suggest that cells present in the media and
adventitia are the predominant source of O2-
in both uninjured and injured vessels.
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To determine which cells present in the media at day +14 may contribute to O2- production, immunohistochemistry and electron microscopy were performed on perfusion-fixed LADs. As previously reported,9 10 32 33 the predominant cell types present were elongated smooth muscle cells (myofibroblasts) and fibroblasts (not shown). Very few lymphocytes or monocytes were observed at this time, as assessed by HCD-12 antibodies and electron microscopic ultrastructure. Thus, smooth muscle cells and fibroblasts appear to be the primary sources of O2-.
The effect of vitamin treatment on the production of
O2- by the uninjured RCA and injured LAD was
then analyzed. Based on the data presented in Fig 4, the following major findings are
important. (1) In pigs that received no vitamins (Fig 4A, LAD), injury
stimulated a 2.5-fold increase in O2-
production by the LAD compared with the uninjured LAD (Fig 1, P=.001). The results for the injured LAD were also compared
with the uninjured RCA of the same animals (Fig 4A). Although uninjured
LAD and RCA had the same O2-
production, a 2.5-fold increase in O2-
production was found in the injured LAD. (2) Analysis
of the treatment effects on RCA O2-
production by one-way ANOVA indicated a highly significant
effect (P=.02 versus control). O2-
production by the RCA in groups C, E, and C+E showed
significant decreases of 42%, 36%, and 45%, respectively, compared
with the RCA in the control (P=.01 versus control). (3)
There were no significant differences in RCA
O2- production among any of the
treatments (group C versus E, C versus C+E, E versus C+E,
P>.1). (4) Analysis of the treatment effects on LAD
O2- production also indicated a
significant effect (P=.01). Specifically, the decrease in
O2- production on a percent basis was
48% in group C (Fig 4B versus 4A, P=.001), 41% in group E
(Fig 4C versus 4A, P=.04), and 54% in group C+E (Fig 4D
versus 4A, P=.001). (5) There were no significant
differences in LAD O2- production
among any of the treatments (group C versus E, C versus C+E, E versus
C+E, P>.1). (6) The magnitude of the decrease in
O2- production for the RCA and LAD on
a percentage basis did not differ significantly among the treatments.
On an absolute basis (cpm/µg protein), however, the decrease in LAD
O2- production was significantly
greater (2.9-fold greater, P=.001). (7) The effectiveness
of treatment to decrease O2-
production was the same for the RCA and LAD: C+E >C >E (52%,
47%, and 40% reductions, respectively). In summary, these data
demonstrate significant decreases in O2-
production of uninjured RCA and injured LAD after treatment
with vitamin C or vitamin E, with the greatest effect observed for the
combination of vitamins C and E.
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To evaluate whether the effect on O2- production by treatment was related to vitamin concentrations, Pearson correlation coefficients were calculated for the levels of vitamins C and E in plasma and lymphocytes against O2- production by the RCA and LAD from each animal. A significant correlation was obtained for lymphocyte vitamin E concentration and LAD O2- production (P=.025) but not for RCA O2- production. No significant correlation was observed for LAD O2- production and plasma vitamin C or E concentration or for lymphocyte vitamin C concentration.
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Discussion |
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The present study of balloon-injured porcine coronary arteries demonstrates three important findings regarding oxidative stress and the vessel response to injury. First, there is a 2.5-fold increase in O2- production by the injured vessel segment 14 days after injury. Second, this increase appears to be caused by alterations in O2- production by cells present in the media and neointima, because removing the endothelium had little effect. Finally, administration of vitamin C or vitamin E, alone or in combination, significantly decreased O2- production.
Several lines of evidence suggest that the effect of the vitamins is due, in part, to modification of vessel redox state. There was a correlation between the lymphocyte vitamin E concentrations (an index of tissue vitamin E levels) and vessel O2- production. There was also a correlation between the inhibition of ex vivo LDL oxidation observed in our previous study10 (conjugated diene lag phase: group C+E>E>C) and the extent of inhibition of O2- production by vessels in the present study (lucigenin chemiluminescence: group C+E>C>E). Finally, there was a trend toward a greater effect of the combination of vitamins C and E than either vitamin alone on O2- production for both LAD and RCA. The enhanced effect of the combination was anticipated because of the ability of vitamin C to augment the antioxidant effects of vitamin E in biological systems.1 These findings suggest that the antioxidant effects of vitamins C and E account, at least in part, for the changes in O2- production. Of note, the tissue concentrations (measured by lymphocyte levels) of vitamin E were significantly higher proportionately than the plasma concentrations, indicating the importance of tissue uptake and binding for this vitamin.
Cell types present in the injured vessel wall that may contribute to increased O2- production include endothelial cells, neutrophils, macrophages, fibroblasts, and VSMCs. In the first 3 days after pig coronary artery injury, many neutrophils are present, but these disappear within 7 days32 33 and are unlikely to account for the O2- production measured at 14 days in the present study. Immunohistochemistry and electron microscopic analysis of the injured vessel at 14 days showed no neutrophils or lymphocytes. However, small numbers of monocytes/macrophages are present in the neointima and adventitia.33 The effect of adventitia was not investigated because it was dissected free to remove blood cells. Endothelial cells appear to contribute little to O2- production, as shown by the results of endothelial denudation. Thus, the dominant source of O2- appears to be cells in the media and neointima, including smooth muscle cells and fibroblasts. The cell type that migrates and proliferates at the site of internal elastic lamina rupture to form the neointima may be described as a myofibroblast.25 Because this is the only cell type that we have observed in the injured segment that is not present in the uninjured segment, it is a likely source of O2- production. In addition, changes in the function of smooth muscle cells and fibroblasts may be present in the injured segment that contribute to O2- production. Future experiments using techniques that simultaneously identify cell type and O2- production will be necessary to prove which cells contribute the most to O2- production.
Treatment with vitamins C and E decreased O2- production in the injured LAD, as expected. We believe that decreased O2- production may be due to the ability of the antioxidants to inhibit O2- production directly via their antioxidant properties and indirectly by several mechanisms. First, vitamins C and E may have induced antioxidant mechanisms, such as superoxide dismutase or glutathione peroxidase. Second, there may have been effects of vitamins on the extent and magnitude of white blood cell adhesion to the damaged vessel and transmigration. This possibility is suggested by studies showing that leukocyte adhesion to endothelium after ischemia-reperfusion is decreased in animals that received vitamin E supplementation34 and that leukocyte adhesion to endothelium stimulated by oxidized LDL is diminished by supplementation with vitamin C.35 Finally, vitamins C and E may alter the expression of proteins responsible for production of O2-. Several intracellular mechanisms may produce excessive O2-, including mitochondrial respiration, intracellular and extracellular oxidases, and various mono-oxygenases.36 Likely candidates to explain increases in cell O2- production after vessel injury include mitochondrial oxidative phosphorylation defects as proposed by Wallace,37 growth factor–regulated O2--producing enzymes such as NADH and NADPH oxidases,22 xanthine oxidase,38 and both secreted and cytosolic forms of phospholipase A2.39 40 We have shown that phospholipase A2 activity is stimulated in cultured VSMCs by oxidative stress and that this leads to proto-oncogene expression, possibly promoting smooth muscle cell proliferation.40 41 42 Griendling and colleagues22 recently demonstrated that NAD(P)H oxidase was highly regulated in cultured VSMCs by angiotensin II. Future work will be necessary to determine the relative contributions of these various O2--generating mechanisms to the increased O2- levels (and decreased levels in vitamin-treated animals) demonstrated in the present study.
We anticipated that endogenous antioxidant mechanisms would
be more than adequate in uninjured vessels and that supplemental
antioxidants should have no effect. In fact, we observed an 40%
decrease in O2- production by the
uninjured RCA in response to vitamin treatment. It is likely that the
same mechanisms as responsible for reduction in
O2- production by the LAD were also
responsible for decreased O2-
production in the RCA. Nonetheless, the finding of decreased
RCA O2- production in animals treated
with vitamin C or E suggests that endogenous antioxidant
mechanisms are "set" at a level that allows significant
O2- production. This concept is
supported by findings from Rajagopalan et al,26 who showed
that infusion of liposome-encapsulated superoxide dismutase was
associated with a decrease in vascular O2-
production. Alternatively, vitamins C and E may have effects in
addition to their role as antioxidants that may modulate the expression
of proteins responsible for O2-
production.
The reduction in O2- production in
animals receiving only vitamin C is surprising. Pigs are able to make
their own vitamin C, and thus, we anticipated that dietary supplements
would have minimal effect on circulating or tissue vitamin C levels. In
fact, animals that received vitamin C alone exhibited a 100% increase
in plasma vitamin C and a 50% increase in lymphocyte vitamin C. Thus,
despite endogenous vitamin C production,
supplementation with 500 mg/d caused significant increases in
plasma and lymphocyte vitamin C concentrations (except for lymphocytes
in group C+E). We observed that vitamin C concentrations in both plasma
and lymphocytes were lower (50% reduction) in animals that also
received vitamin E compared with vitamin C alone. This was not due to
an effect of vitamin E on measurement of vitamin C, as determined by
addition of exogenous vitamin E to the assay (data not shown). A
possible explanation is that excretion of vitamin C is increased when
vitamin E is coadministered. Whether vitamin C levels in lymphocytes
adequately reflect vessel concentrations and/or metabolism
of exogenously supplied vitamin C is unknown.24 43 The
techniques for determining vitamin levels in tissue are complex,
determined by tissue processing, tissue metabolism, and
extraction. A limitation of the present study was our inability to
measure the vessel levels of vitamins C and E because of small amounts
of tissue and the difficulty in solubilizing the muscular
coronary arteries. Thus, interpretations based on extrapolating
from plasma and lymphocyte vitamin concentrations to tissue
concentrations may be imprecise.
The findings of the present study provide insights into previously reported effects of antioxidants on vessel morphology and structure in the pig injury model.9 10 Probucol was found to decrease maximal intima thickness.9 In contrast, vitamins C and E caused 20% to 25% increases in lumen area and vessel area without a significant change in maximal intima thickness.10 We interpret these data to show that the primary effect of vitamins C and E is to enhance outward vessel remodeling (promoting vessel enlargement and/or preventing vessel constriction), whereas the primary effect of probucol is to inhibit VSMC proliferation. Probucol has actions in addition to being an antioxidant that may explain its effect on VSMC proliferation, including inhibition of interleukin production and modification of lipoprotein composition.2 3 44 45
The correlation between changes in vessel O2-
production and vessel morphology/structure is complex. Vitamins
C and E in combination had the greatest effect on vessel
morphology10 and O2-
production (Fig 4). Vitamin C alone or vitamin E alone had
little effect on vessel morphology. However, vitamin C alone and
vitamin E alone had significant effects on O2-
production, nearly equivalent to the combination of vitamins C
and E. Thus, the failure of vitamin C or vitamin E alone to promote
vessel remodeling cannot be explained by an inability to decrease
O2- production. A possible explanation
for this discrepancy may be that vitamin C and E alone do not have
effects on other reactive oxygen species (eg,
H2O2 and OH-) that are equivalent
to the combination. Alternatively, the beneficial effect of the
combination of vitamins C and E may be due to changes in cellular
mechanisms other than redox state. Finally, there may be effects on
cells that are too subtle for us to measure with the techniques used in
this study. One example would be a change in
endothelial cell redox state, which would have only a
small effect on total O2-
production (see Fig 3) but might have important functional
consequences for endothelium-dependent
vasorelaxation.15 16
It is tempting to extrapolate the present data to humans. However,
extrapolation to humans is complicated by differences in age (weanling
pigs were studied), lipid status (cholesterol was 70
mg/dL), and vitamin metabolism. The changes in
vitamin E levels achieved by supplementation were smaller than those
observed in humans. Baseline vitamin E was 200 µg/dL and
increased to 475 µg/dL. In healthy humans (average age, 36
years), baseline vitamin E is 1000 µg/dL and increases to 2000
µg/dL after 800 IU/d for 8 weeks.46 Finally, it
is important to note that although vitamin C is an essential vitamin in
humans, pigs synthesize vitamin C from dietary
sources.24
Several recent studies have yielded conflicting results regarding the
effects of antioxidant vitamins and probucol to limit
atherosclerosis and to prevent restenosis after
angioplasty. In two large population studies, individuals who consumed
400 IU/d of vitamin E had a 40% reduction in
cardiovascular events.4 5 In the CLAS
study, there was decreased progression of coronary artery
disease in patients receiving vitamin E.47 More recently,
the CHAOS trial, which randomized patients with known coronary
artery disease to placebo or vitamin E (400 or 800 IU/d), showed a 60%
reduction in nonfatal myocardial infarction.48 A small
study of restenosis based on exercise thallium testing showed a
benefit of vitamin E in limiting restenosis.13
Three studies have shown a beneficial effect of probucol on
restenosis after angioplasty,14 49 50 and one
study was negative.51 Lee et al,50 in the
PART study, found a significant decrease in restenosis in
patients who received probucol, but this benefit was observed only in
patients who received 30 days of pretreatment. Watanabe et
al49 found a similar benefit of probucol administered 7
days before PTCA. Tardif et al,14 in the MVP study
(presented only in abstract form), compared probucol and
multivitamins (ß-carotene, vitamin E, and vitamin C) alone or in
combination with 28 days of pretreatment. They found a significant
decrease in restenosis with probucol but not with the
multivitamins. Finally, O'Keefe et al,51 in the APPLE
study, found no benefit of lovastatin and probucol together
on clinical events, exercise thallium, or angiographic
restenosis. It should be noted that probucol was given 2 days
before to 1 day after PTCA in this last study. The MVP study is the
only study to compare antioxidant vitamins with probucol directly. This
study was well designed in that treatment was initiated 28 days before
angioplasty. The reasons why antioxidant vitamins failed to limit
restenosis in the MVP study are not clear, but concomitant
treatment with ß-carotene may have exerted a harmful effect.
Alternatively, longer follow-up may be required before beneficial
effects of antioxidant vitamins can be observed, as suggested by the
CHAOS trial, in which differences with vitamin E were not observed for
>1 year.48 Because the MVP study has been
presented only in abstract form, relating results of that study
to the present study is difficult. There are two likely reasons why
the MVP study showed no benefit for antioxidant vitamins and the
present animal study showed a beneficial effect. First, in the MVP
study, the coadministration of ß-carotene with vitamins C and E may
have had a deleterious effect that masked the potential beneficial
effects of vitamins C and E. Second, the presence of
hyperlipidemia and established coronary
atherosclerotic disease in the MVP patients clearly differs from the
normolipemic, normal coronary arteries of the pigs in the
present study. Future studies with vitamin C and E alone will be
necessary to resolve questions of their efficacy in limiting
restenosis after angioplasty. Nonetheless, the present
study is the first to show increased O2-
production in injured vessels and to demonstrate that
antioxidant vitamins reduce O2-
production. These results suggest that further understanding of
the mechanisms by which O2- is produced after
injury may provide insight into therapies to limit restenosis
after balloon angioplasty and progression of
atherosclerosis.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This work was supported by grant HL-44721 from the National Institutes of Health to Dr Berk. Dr Berk is an Established Investigator of the American Heart Association. We thank Jody Miyashiro and Marshall Corson for helpful discussions and critical reading of the manuscript. We acknowledge the excellent assistance of Robert Redden, Tim Peterson, and Gus Cippolla in performing many of the assays.
Received April 2, 1997; revision received June 23, 1997; accepted June 26, 1997.
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References |
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W.-G. Li, F. J. Miller Jr, M. R. Brown, P. Chatterjee, G. R. Aylsworth, J. Shao, A. A. Spector, L. W. Oberley, and N. L. Weintraub Enhanced H2O2-Induced Cytotoxicity in "Epithelioid" Smooth Muscle Cells : Implications for Neointimal Regression Arterioscler. Thromb. Vasc. Biol., June 1, 2000; 20(6): 1473 - 1479. [Abstract] [Full Text] [PDF]
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P. Sinnaeve, O. Varenne, D. Collen, and S. Janssens Gene therapy in the cardiovascular system: an update Cardiovasc Res, December 1, 1999; 44(3): 498 - 506. [Abstract] [Full Text] [PDF]
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 M. Jeserich, T. Schindler, M. Olschewski, M. Unmussig, H. Just, and U. Solzbach Vitamin C improves endothelial function of epicardial coronary arteries in patients with hypercholesterolaemia or essential hypertension--assessed by cold pressor testing Eur. Heart J., November 2, 1999; 20(22): 1676 - 1680. [Abstract] [PDF]
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T. Saldeen, D. Li, and J. L. Mehta Differential effects of {alpha}- and {gamma}-tocopherol on low-density lipoprotein oxidation, superoxide activity, platelet aggregation and arterial thrombogenesis J. Am. Coll. Cardiol., October 1, 1999; 34(4): 1208 - 1215. [Abstract] [Full Text] [PDF]
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M. R. Brown, F. J. Miller Jr, W.-G. Li, A. N. Ellingson, J. D. Mozena, P. Chatterjee, J. F. Engelhardt, R. M. Zwacka, L. W. Oberley, X. Fang, et al. Overexpression of Human Catalase Inhibits Proliferation and Promotes Apoptosis in Vascular Smooth Muscle Cells Circ. Res., September 17, 1999; 85(6): 524 - 533. [Abstract] [Full Text] [PDF]
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M. J. Pollman, J. L. Hall, and G. H. Gibbons Determinants of Vascular Smooth Muscle Cell Apoptosis After Balloon Angioplasty Injury : Influence of Redox State and Cell Phenotype Circ. Res., January 22, 1999; 84(1): 113 - 121. [Abstract] [Full Text] [PDF]
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 R. Ross Atherosclerosis -- An Inflammatory Disease N. Engl. J. Med., January 14, 1999; 340(2): 115 - 126. [Full Text] [PDF]
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K. Miwa, A. Igawa, K. Nakagawa, T. Hirai, and H. Inoue Consumption of vitamin E in coronary circulation in patients with variant angina Cardiovasc Res, January 1, 1999; 41(1): 291 - 298. [Abstract] [Full Text] [PDF]
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A. M. Zafari, M. Ushio-Fukai, M. Akers, Q. Yin, A. Shah, D. G. Harrison, W. R. Taylor, and K. K. Griendling Role of NADH/NADPH Oxidase–Derived H2O2 in Angiotensin II–Induced Vascular Hypertrophy Hypertension, September 1, 1998; 32(3): 488 - 495. [Abstract] [Full Text] [PDF]
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