© 1995 American Heart Association, Inc.
Articles |
Combination of Vitamins C and E Alters the Response to Coronary Balloon Injury in the Pig
From the Departments of Medicine (Cardiology Division, Andreas Gruentzig Cardiovascular Center) (G.L.N., R.A.R., S.R.S., S.B.K.) and Pathology (D.S.S., M.B.G.), Emory University School of Medicine, Atlanta, Ga, and the Department of Medicine (Cardiology Division), University of Washington School of Medicine (B.C.B), Seattle.
Correspondence to Bradford C. Berk, MD, PhD, Cardiology Division, Department of Medicine, RG-22, University of Washington, Seattle, WA 98195.
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Abstract Restenosis is the major limitation of the long-term success of percutaneous transluminal coronary angioplasty. The process of restenosis involves repair of vascular injury and remodeling of vessel architecture. Therapeutic interventions that improve vascular function may therefore be beneficial in the treatment of restenosis. Antioxidants such as probucol and vitamins C and E have proved effective in improving endothelial function in hypercholesterolemia, inhibiting lipid accumulation in animal models of atherosclerosis, and decreasing cardiovascular mortality in humans. Forty-two female domestic swine were divided into four study groups: control (n=12); vitamin C (500 mg/d, group C, n=9); vitamin E (1000 U/d, group E, n=10); and vitamins C and E (500 mg/d + 1000 U/d, group C + E, n=11) before oversized balloon injury of the left anterior descending and circumflex coronary arteries. Vitamins were administered 7 days before balloon injury and continued until the swine were killed 14 days after injury. Significant differences in morphometric parameters were present only in group C + E, with increases in vessel and lumen area in the segment with maximal injury. Although there was no decrease in intima area or in maximal intima thickness, the ratio of intima area to vessel area was significantly reduced, consistent with a positive effect in group C + E. Graphic analysis of the relationship between initial vessel injury (using internal elastic lamina fracture length/lumen perimeter) and vessel response to injury (using intima area/vessel area) for all segments showed improved indices for group C + E (P<.005). The beneficial effect of vitamins correlated with changes in lipid redox state. Low-density lipoprotein (LDL) thiobarbituric acid–reactive substances showed an
70%
decrease in all treatment groups, and the lag phase for LDL-conjugated
diene formation was significantly increased, with group C + E>group
E>group C. The combination of vitamins C and E improved vascular
response to injury because of an apparent beneficial effect on vascular
remodeling. The fact that the combination of vitamins C + E was
better than vitamin E or vitamin C alone is consistent with the ability
of vitamin C to improve the antioxidant effect of vitamin E, suggesting
that the improved vessel response was due to a change in redox state.
This study suggests an important role for oxygen radicals in the
vascular response to injury and suggests that vascular remodeling and
intimal proliferation are important to the restenotic process.
Key Words: antioxidant • restenosis • reactive oxygen species • vascular remodeling
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Introduction |
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Restenosis after percutaneous transluminal coronary angioplasty is the major limitation of the long-term success of this procedure.1 The primary mechanisms proposed for restenosis are proliferation of vascular smooth muscle cells (VSMCs) and formation of a neointima that reduces the vessel lumen. Despite numerous trials, no pharmacological intervention has significantly reduced the frequency of restenosis since the inception of angioplasty 15 years ago.2 3 4 5 New mechanical approaches to angioplasty, including atherectomy, laser, and rotoblater, have also failed to reduce restenosis.6 7 8 9 10 11 Preliminary data suggest that intravascular stents may be associated with decreased restenosis rates12 13 but at the cost of a retained foreign body, increased risk of vascular complications, and increased cost of initial hospitalization. The failure of these approaches to limit restenosis indicates that our knowledge of the underlying biology of restenosis is incomplete and that other processes besides smooth muscle cell proliferation and neointima formation must also be critical. In fact, recent analyses of the relation between intima mass and lumen diameter have failed to show a significant correlation.14 15 16 When combined with recent data that demonstrate a low level of smooth muscle cell proliferation in restenotic tissues,17 these studies indicate that processes in the vessel wall besides smooth muscle cell proliferation are critical to determining the lumen diameter at follow-up. These processes may be considered together as "vessel remodeling," which may involve alterations in matrix deposition, adventitial growth and repair, and changes in endothelial regulation of vessel tone and growth.
The finding that nitric oxide, a critical regulator of vascular function, is inactivated by oxygen radicals suggests that the vessel response to injury (ie, "remodeling") may be altered by vessel redox state.18 Thus, an evolving concept in the pathogenesis of vascular injury and atherogenesis is the role of oxygen radicals and oxidative stress.19 Oxidized `low-density lipoprotein (LDL) contributes to foam cell formation and has been found within atherosclerotic plaque. The importance of oxidative stress in the initiation of atherosclerosis has been demonstrated in animal models. One of the most dramatic examples was the ability of the antioxidant drug probucol to limit atherogenesis in the Watanabe heritable hyperlipidemic rabbit.20 21 Other investigators22 23 have shown that antioxidants can limit neointima formation in response to balloon injury in the hypercholesterolemic rabbit model. On the basis of these results, we have already tested the ability of probucol to limit neointima formation in a swine model of coronary artery balloon injury.24 We showed that probucol (2 g/d) significantly reduced neointima thickness 14 days after balloon injury, suggesting that oxygen radicals are important in this process. Recently, it has been shown that intake of dietary supplements of vitamin E is associated with a significantly decreased risk of coronary heart disease in men and women.25 26 In addition, DeMaio et al27 showed a beneficial effect of vitamin E supplementation in patients 6 months after angioplasty (assessed by exercise testing).
In the present study, we tested the hypothesis that the antioxidant vitamins C and E would favorably alter the vessel response to balloon injury in the swine coronary. The swine model offers specific advantages over other animal models for the study of restenosis because of the similarities with the human coronary circulation, spontaneous development of atherosclerosis, and histological response to vascular injury.28 29 30 Based on our previous experience, important parameters chosen for analysis in the present study were analysis of the left anterior descending (LAD) and circumflex arteries separately because of the greater injury in the smaller circumflex, measurement of circulating lipid redox state, and development of a method to analyze morphological changes present in all segments of the injured artery in contrast to previous techniques that used only the segment with greatest injury. Our findings show a significant reduction in the response to injury with a combination of vitamins C and E. Because this combination of vitamins was also the most effective in altering lipid redox state, these data suggest that the improved vessel response was a consequence of inhibiting oxygen radical formation.
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Animals
Forty-two juvenile female domestic swine were used in this study (weight, 20 to 34 kg; mean, 26.4±3.9 kg). Pigs were treated starting 1 week before coronary balloon injury (day -7) with vitamin C 500 mg/d (group C, n=9); vitamin E 1000 U/d (group E, n=10); both (group C + E, n=11); or neither (control, n=12). 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 U/kg vitamin E, and no vitamin C) was given to all animals.
All experimentation and animal care conformed to the National Institutes of Health (NIH) and American Heart Association guidelines for the care and use of animals. The Emory University Animal Care and Use Committee approved the study.
Experimental Protocol
After 7 days of vitamin supplements (day 0), the animals
underwent coronary overstretch balloon injury, as previously
described.24 28 Briefly, the animals were sedated with a
combination of ketamine (25 mg/kg; Aveco), acepromazine (1.1 mg/kg;
Aveco), and atropine (0.6 mg/kg; Gensia Pharmaceuticals) administered
by intramuscular injection. After an intravenous line was established,
each animal received intravenous Brevitol (10 mg/kg or to effect; Eli
Lilly) to allow endotracheal intubation. A continuous intravenous
infusion of Ringer's solution was maintained to provide hydration
during the procedure. Each animal was ventilated with oxygen (2 L/min),
nitrous oxide (2 L/min), and isofluorane 1% (2 L/min; Anaquest) to
maintain adequate anesthesia as determined by the absence of the limb
withdrawal reflex. Throughout the procedure, electrocardiograms and
intra-arterial pressure were monitored. No significant changes in blood
pressure were observed during the procedure.
An 8F introducer was placed in the right femoral artery through a cutdown, and coronary angiography was performed using an 8F hockey stick guiding catheter. Intracoronary nitroglycerin (200 µg) was administered before each contrast injection. On average, each animal received two injections of nitroglycerin (one before the baseline angiogram recorded before balloon injury and another injection before the final angiogram). There was no significant systemic effect, as measured by femoral artery mean arterial pressure, which was unchanged during the procedure. Both angiograms were performed after the dilation system (steerable wire and balloon catheter) was removed from the guiding catheter and the animal. All angiographic measurements were determined with end-diastole frames. Angiography was performed only during initial injury to verify successful injury and to confirm that there were no differences in the extent of initial injury among groups. We did not perform follow-up angiography at 14 days because previous studies failed to show any significant differences in vessel parameters.24 31
Coronary injury was achieved by deliberate stretch of the target vessel using 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 and circumflex arteries. Inflations were separated by 1-minute intervals to allow coronary perfusion. After repeat coronary angiography, the catheters were removed and the cutdown was repaired. Nitroglycerin ointment (1") was applied topically, and the animals were allowed to recover.
Vessels were harvested 21 days after the initiation of treatment (day 14). To harvest vessels, the animals were injected with intravenous heparin (200 U/kg) and were 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. For histological analyses, the coronary system was perfusion-fixed with 10% buffered formalin for 15 minutes at physiological pressure (100 mm Hg). These procedures were carried out immediately after the animals were killed in the animal surgery room.
Angiographic Analysis
The angiograms performed on the day of injury (day 0) were
analyzed with a computer-based system. The cine frames were converted
to computer images and digitized with a Macintosh IIcx computer using a
high-resolution video digitizing board. The images were processed using
NIH IMAGE software (IMAGE processing
software developed by Wayne Rasband, NIH, Bethesda, Md, and modified by
J. Larry Klein, Emory University School of Medicine, Atlanta, Ga). An
experienced cardiac angiographer blinded to the treatment groups
analyzed the angiographic data. Measurements included the baseline
diameter of the vessel segment before balloon injury, the
balloon-to-artery ratio, and the final diameter of the dilated segment
immediately after injury. All measurements were corrected with the
guiding catheter as the reference object.
Vitamin, Lipid, and Lipid Oxidation Measurements
Blood samples for determination of vitamin levels, serum
cholesterol, and plasma redox state (thiobarbiturate acid–reactive
substances [TBARS] and conjugated diene formation) were collected
at day -7 (before vitamin supplements), day 0 (day of balloon injury),
and day 14 (vessel harvest). Blood from individual pigs was collected
in tubes containing EDTA (1 mg/mL), and butylated hydroxytoluene (4.4
µg/mL) was also added to blood collected for vitamin assays. Plasma
was prepared by low-speed centrifugation at 4°C. LDL was prepared by
stepwise ultracentrifugation in a density range of 1.020 to 1.060
g/mL.32 EDTA-containing LDL preparations were stored at
4°C in the dark up to 1 week until assayed. Each LDL preparation was
dialyzed against degassed 0.01 mol/L sodium phosphate buffer, 0.15
mol/L NaCl, pH 7.4, for at least 24 hours and stored until use at 4°C
under nitrogen in the dark for 
24 hours.
LDL preparations were diluted with EDTA-free, oxygen-saturated 0.01 mol/L NaCl, pH 7.4. Oxidation was initiated by addition of freshly prepared cupric sulfate solution to LDL at 37°C. The final concentrations were 0.25 mg LDL protein/mL solution and 50 µmol/L cupric sulfate.33 Oxidation rate was followed in a spectrophotometer by continuously recording of the increase in absorption at 234 nm, which is characteristic of conjugated lipid hydroperoxides. The lag phase was defined as the time between the start of oxidation (T=0) and the x intercept of a least-squares regression of the oxidation curve during the propagation phase. LDL oxidation was also measured by the TBARS method as modified by Babiy et al.34 In this assay, 50 µL LDL solution containing 100 µg protein was mixed with 150 µL 20% trichloroacetic acid and 150 µL 0.67% thiobarbituric acid. After incubation at 100°C for 1 hour, the samples were centrifuged for 15 minutes, and 200 µL supernatant was transferred to a 96-well microplate. Absorbance at 540 nm was measured in the samples with an automatic microplate reader. Freshly prepared malonaldehyde tetramethylacetal solution was used as a standard.
-Tocopherol was determined by high-performance liquid chromatography
(HPLC), as previously described.35 The HPLC system
consisted of a Milton Roy Consta Metric pump, a 3.9-mmx30-cm column of
µ-Bondapak C18, a guard column packed with µ-Bondapak C18 Corasil,
and a fluorescence spectrophotometer (Hewlett Packard 1046A). The
mobile phase was water in methanol (2:98 by vol), and the flow rate was
1.0 mL/min. The excitation wavelength was set at 292 nm, and the
emitted fluorescence was measured at 333 nm. Peak areas were measured
electronically and calculated with an external standard.
Plasma ascorbic acid concentration was quantified by use of a colorimetric phosphotungstic acid assay.36 Cholesterol was determined with a colorimetric-enzymatic method on the Olympus Au 5000 Clinical Chemistry Analyzer (Olympus Corporation).
Tissue Analysis
The injured coronary segments of the LAD and circumflex arteries
were located with the guidance of the coronary angiograms and dissected
in block from the heart. Serial 4-mm sections were processed and
embedded in paraffin. Cross sections (4 µm thick) were stained with
hematoxylin-eosin, Verheoff-van Gieson's, and Masson's trichrome
stains. The specimens were then analyzed by an experienced
cardiovascular pathologist (blinded to the treatment group) and
evaluated for the presence of fracture of the internal elastic lamina,
intimal hyperplasia, luminal encroachment, medial dissection, and
presence of luminal and/or intramural thrombi.
Morphometric Analysis
Two approaches to morphometry were used. (1) The site of maximal
injury as defined by maximal intimal area was determined and vessel
parameters were measured in this section. (2) Proportionate vessel
morphometric parameters (eg, injury response/vessel area or injury
response/lumen area) in all segments demonstrating injury were
determined. Numerous absolute measurements of smooth muscle cell
proliferation have been used in animal models of restenosis, but none
account for changes in absolute vessel size resulting from vessel
remodeling. Stimulated by the work of Bonan et al,37 we
determined whether comparison of an initial injury index relative to a
response to injury index would be more meaningful in assessment of
vessel response to treatment after overstretch balloon injury in
porcine coronary arteries. In particular, we hypothesized that use of
proportionate vessel morphometric parameters would improve subsequent
statistical analysis. Morphometric analysis was performed in
all injured segments of each vessel (3 to 5 per vessel) by use of a
computer-interfaced image analysis system (Optimas Bioscan 2,
Thomas Optical Measurement System, Inc). A clear advantage of this
technique over techniques that consider only the segment showing
maximal intimal response is that more data points are obtained from
each vessel. Several morphometric descriptors were measured (Fig 1
): lumen perimeter (LP), vessel perimeter (VP,
circumference of the external elastic lamina), maximal intima thickness
(MIT, line between the lumen and outermost point of the
neointima), fracture length (FL, line between the two
fracture points of the internal elastic lamina), LA (area within the
vessel lumen), intima area (IA, area occupied by the
neointima), and vessel area (VA, area within the external
elastic lamina). From these measurements, we also calculated the
residual lumen (RL) defined as LA/(IA+LA). Several different
measurements of FL were performed: traced in the luminal border, along
the external elastic lamina border, and through the media. Both
straight lines and curved lines were compared. The coefficients of
variation of these different measurements were very similar, indicating
that reproducibility was excellent. The best correlation with IA was
achieved by measuring FL through the media as a straight line. The
first analysis was to determine the correlation of these
descriptors with a standard measurement of response to vascular injury,
the IA. A subgroup of eight control animals was used for this
analysis, including both LAD and circumflex arteries. The index of
initial injury that correlated best with intimal area was FL/LP
(R2=.642). The next analysis was to
determine the measurement of vessel response to injury that yielded the
best correlation with FL/LP as an index of the extent of initial
injury. Vessel responses to injury that were compared included MIT,
1-RL, IA/VA, IA/(VA-LA), and intima/media thickness. The
R2 values for the linear regression analysis
of each parameter relative to FL/LP were 0.13, 0.65, 0.75, 0.71, and
0.74, respectively (data not shown). Thus, the best relation was
achieved with IA/VA (R2=.75). Notably, such
standard measures as MIT yielded very poor correlation
(R2=.13). The poor correlation between MIT and
the initial injury is similar to the results reported by others for the
relation between MIT and lumen diameter.14 15 16 Based on
this analysis, all subsequent comparisons of injury and response to
injury were performed with FL/LP as an index of initial vessel injury
and IA/VA as an index of the response to injury. In addition, to gain
insight into the relation between initial injury and LA, we compared
FL/LP and LA. The fact that ratios of parameters gave better
correlations than individual morphological descriptors was expected on
the basis of the use of multiple segments where the extent of initial
injury may have differed significantly. With these measurements,
indices of injury (FL/LP) and vessel response to injury (IA/VA) were
used to generate linear regression lines (FL/LP versus IA/VA) for each
of the four experimental groups, as described in the "Results"
section. No significant difference was noted in the use of this
analysis for LAD compared with circumflex arteries. However, the
circumflex artery exhibited more variability and hence a poorer
correlation.
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Materials
HAM-55 antibody and all other reagents were obtained from Sigma
Chemical Co. Chemicals were dissolved in 50 mmol/L phosphate buffer (pH
7.8) and added into Krebs-HEPES buffer (pH 7.4).
Statistical Analyses
Pathologists and fellows responsible for morphology analyzed all
experiments in a blinded fashion. Data are reported as mean±SEM. We
compared morphometry data by a two-factor 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 Fisher's Exact Test. Baseline swine
data were determined by a one-factor repeated-measures ANOVA (treatment
groups). Statistics were computed with SUPERANOVA (Abacus
Concepts). A value of P<.05 was considered significant. To
compare differences in the slopes of graphical comparisons, linear
regression was performed. The differences were then calculated with
multiple t tests, and a Bonferroni correction was applied to
adjust the alpha level for the total number of t tests
performed.
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Group Characteristics
Table 1
lists the characteristics of the swine
studied. There were no significant differences in animal weight among
the groups at the time of balloon injury. Three swine died during the
initial procedure. One death occurred in each group except the control
group. All deaths were attributed to acute thrombosis of a ballooned
coronary artery. These animals were excluded from subsequent
analysis.
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Serum Cholesterol and Vitamin Levels
Fig 2A illustrates the total cholesterol levels in
the four groups of animals. Total serum cholesterol was unchanged
throughout the study period in all groups. Serum vitamin C levels (not
shown) also did not change, despite the vitamin C supplements given to
animals in groups C and C + E. Because the kidneys rapidly excrete
vitamin C38 39 and pigs synthesize vitamin C
continuously,40 it is likely that the measurements (made 8
to 10 hours after vitamin C supplements) failed to detect the vitamin.
Previous workers39 40 41 have also failed to show significant
increases in plasma vitamin C levels with dietary supplementation. In
contrast, animals that received vitamin E supplements (groups E and
C + E) showed almost a fourfold increase in serum vitamin E levels
after both 1 and 3 weeks of dietary supplements (Fig 2B,
P=.0001), demonstrating that the animals were consuming the
supplements.
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Angiographic Analysis
Angiographic analysis was performed with a computer-based
system to determine the degree of vessel stretch, measured as the ratio
of artery diameter during and after balloon inflation to diameter
before balloon inflation. The mean LAD artery diameter for all animals
was 3.10±0.10 mm before, 3.46±0.03 mm during, and 3.26±0.07 mm after
(n=14, P<.05 versus before) balloon inflation. Thus, the
degree of stretch was 11.2% during and 5.1% after inflation. The mean
circumflex diameter for all animals was significantly smaller
(2.69±0.13 mm before) than the LAD artery diameter. However, the
diameters during (3.42±0.02 mm during) and after (3.15±0.08 mm after,
n=13, P<.05 versus before) balloon inflation were similar
to the LAD artery diameter. Therefore, compared with the LAD, the
circumflex underwent much greater stretch, 27.5% during and 17.1%
after inflation. This larger stretch was associated with greater
injury, as shown below. There were no significant differences in the
extent of vessel dilation in the treatment and control groups (Table 1
), suggesting that the 1-week pretreatment with vitamins did not
measurably alter the vessel response to balloon injury. Because of
previous findings in this model24 28 and to decrease cost,
angiography was not performed at the time of vessel harvest.
Histopathologic Analysis
Histopathologic analysis was performed on all injured segments
(three to five per vessel). As previously demonstrated, the internal
elastic lamina ruptured, with neointima growth replacing
the disrupted media 2 weeks after injury. The neointima
consisted of smooth muscle cells, loose connective tissue, and
proteoglycans, as identified by light microscopy and
immunohistochemistry.24 28 31 Only rare macrophages were
identified in the neointima by the HAM-55 antibody (not
shown). There were no obvious differences in the histology of the
injured vessels in any treatment group, based on analysis of three
to five sections from each animal by a pathologist blinded to the
treatment groups.
We believe that consistent injury occurred in the treatment and control groups because all vessels included in the analysis demonstrated rupture of the internal elastic lamina and the pathological grading scale that we have previously used24 varied little among groups (not shown). Dissection was present in 100% of the vessels. Because there appeared to be greater injury of the circumflex than the LAD artery in previous studies, we specifically measured the degree of dissection and fracture length of the internal elastic lamina in each vessel. Using these measurements, we calculated an injury index (FL/LP) that was significantly greater (P<.0001, n=21 animals) in the circumflex (0.41±0.02) than in the LAD (0.29±0.02). Organized intramural thrombus that appeared to represent platelet thrombi trapped in the dissected media was noted in 2 of the 84 vessels studied (both vessels were circumflex). The thrombus, which was readily defined histologically, was excluded from all morphometric measurements.
Morphometric Analysis at Site of Maximal IA
Several independent measurements of the vessel response to injury
were performed, and comparisons were made for all injured vessels and
separately for LAD and circumflex arteries. These measurements were all
made at the site of maximal injury determined by the largest IA. As
Table 2 shows, four effects were readily demonstrated
when the combined LAD and circumflex data were analyzed (n=21 animals).
First, the treatment group that differed the most from control was
group C + E. Second, there was no significant difference between
control and treatment groups for IA or MIT. Third, there was a
significant increase in the size of the vessel in groups treated with
vitamins C and E assessed by all measurements (VP, LP, LA, VA, IA/VA,
and RL). Fourth, based on analysis of benefit as an increase in LA
or a decrease in IA/VA, significant benefit was demonstrated only for
group C + E.
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Separate analysis of the LAD and circumflex arteries (not shown) revealed two important findings. First, the response to injury, measured as IA, was significantly greater in the circumflex compared with the LAD artery. Specifically, the IA for each treatment group (LAD/circumflex) was 0.71±0.06/0.87±0.07 (control), 0.82±0.09/1.03±0.09 (group C), 0.86±0.10/1.05±0.14 (group E), and 0.71±0.09/0.94±0.09 (group C + E). Second, despite greater IA in the circumflex, the increases in vessel size in group C + E were similar in the two vessels (group C + E/control for LAD and circumflex, respectively): VP (1.13 and 1.17), LP (1.22 and 1.29), LA (1.33 and 1.49), and VA (1.26 and 1.34). Because the morphometric responses to injury were very similar for LAD and circumflex (especially our primary end point, IA/VA), all analyses were combined for the two vessels. In summary, when measured at the site of maximal response to injury (largest IA), the effect of vitamins C and E was to increase vessel size significantly without altering the amount of neointima present.
The effects of vitamins C + E are shown for elastin-stained vessels
in Fig 3. The left panel shows representative
photomicrographs of the circumflex artery. It is apparent that with
injury (Fig 3B) there is rupture of the internal elastic lamina and
growth of VSMCs to form a neointima. The most impressive
effects were observed for treatment with vitamins C + E, which caused
an increase in vessel size and a relative decrease in the
neointima area to VA (Fig 3C). The right panel in Fig 3
shows that the LAD artery is generally larger than the circumflex and
that the effect of vitamins C + E is more apparent (compare Fig 3B and 3C to Fig 3E and 3F).
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Morphometric Analysis of All Injured Sites
In addition to measurements at the site of maximal injury, we also
analyzed all vessel segments that exhibited any injury using the
proportionate parameters discussed in the "Methods" section. The
relation between the initial injury index (FL/LP) and the subsequent
vessel response to injury (IA/VA) was analyzed by linear regression
(Fig 4). A decrease in slope implies a positive
treatment effect, ie, less IA for a given extent of initial injury. The
treatment groups showed progressively smaller slopes with group
C + E<group E<group C, indicating greatest benefit for group
C + E. Only the combined treatment significantly improved the vessel
response to injury (P<.005). The treatment with vitamin E
showed a trend toward significance (P<.05). A similar
analysis compared initial injury index (FL/LP) and the final LA.
This analysis demonstrated a similar result (not shown): there was
a highly significant increase in LA relative to initial injury in group
C + E.
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In summary, the effect of vitamins C and E on the injured artery
was to increase vessel and lumen size more than to inhibit
neointima growth. This ability to increase vessel size
results in a beneficial response to injury whether the benefit is
analyzed by LA (LA increased 39% by treatment with vitamins C + E),
by response to injury (IA/VA decreased 21% by treatment with vitamins
C + E), or by the relation between initial injury and response to
injury (Fig 4, comparison of FL/LP and IA/VA).
Lipid Oxidation State
To study how lipid oxidation related to the effect of vitamins on
vascular injury, we measured both TBARS and conjugated diene formation
in LDL. A significant change in lipid oxidation was observed after
vitamin supplements (Figs 5 and 6). LDL
TBARS were decreased significantly by the 7 days of treatment before
injury and had decreased by 69±1% at day +14 in animals that received
vitamins C and E, either alone or in combination (Fig 5,
P=.0001 versus control). Conjugated diene formation was
measured only at the time of vessel harvest (day +14). There were
dramatic increases in the time required to conjugate dienes in groups E
and C + E. Lag times were 15, 20, 60, and 120 minutes for the control
group and groups C, E, and C + E, respectively. Thus, all treatment
groups showed significant decreases in oxidation of circulating
LDL.
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Discussion |
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The major finding of this study is that vitamins C and E improve the vascular response to balloon injury, as demonstrated by a larger lumen diameter and decreased IA/VA ratio 2 weeks after injury. This result appears to be due to a beneficial effect on vascular remodeling. The term "remodeling" was chosen to describe the present findings because the vascular response to injury involves not only intimal cell proliferation but also changes in the tissue structure of the media and adventitia.14 42 In the pig coronary artery, the combination of vitamins C and E caused a relative decrease in the amount of intima (IA/VA) for a given amount of injury (FL/LP). However, this was due to increases in lumen area (1.96 versus 2.76 mm2 in control and C + E groups, respectively) and vessel size (3.40 versus 4.42 mm2 in control and C + E groups, respectively), rather than a decrease in IA (0.78 versus 0.82 mm2 in control and C + E groups, respectively).
Remodeling as an Explanation for Effects of Vitamins C and E
Several recent studies43 44 of vessel response to
injury as it pertains to clinical restenosis have suggested that two
measurements of this response should be independently evaluated: the
growth of neointima, as a marker of the biological response
to injury, and the follow-up luminal diameter, as a marker of the
clinical significance of the result. Because it was not possible to
measure cell proliferation in the pig coronary in the present
study, we cannot relate the increase in lumen diameter observed to
changes in smooth muscle cell replication and intima mass. Recent
analyses of the relation between intima mass and lumen diameter have
failed to show a significant correlation between these
variables.14 16 These studies indicate that processes in
the vessel wall besides smooth muscle cell proliferation are critical
to determining the lumen diameter at follow-up. A similar conclusion
has been arrived at by analysis of the response of the vessel to
balloon angioplasty compared with other mechanical interventions
(laser, atherectomy, stent).43 44 These studies have
defined the term "loss index" (acute gain in lumen diameter/late
gain) as a measure of the relative efficacy of any intervention to
prevent restenosis. Most interventions (when used in optimal clinical
settings) have a loss index of 0.5, meaning that for each 1.0 mm of
gain in lumen diameter initially, 0.5 mm is lost at a 6-month
follow-up. The concept of loss index suggests that efforts to create a
larger initial lumen will generate a greater loss in subsequent lumen
diameter. When coupled with recent data that demonstrate a very low
level of smooth muscle cell proliferation in restenotic tissues
obtained by atherectomy,17 it is apparent that other
events in the vessel wall are contributing to restenosis, such as
remodeling.
Although speculative, interpretation of the present findings as a remodeling response is strengthened by three recent studies demonstrating similar morphological results in other balloon injury models.14 15 42 In all studies, there was a correlation between initial injury (initial gain in human studies) and response to injury (measured by IA). However, there was no significant relation between the final lumen diameter ( loss of acute gain in human studies) and IA. In the present study, there was a highly significant correlation between initial injury (FL/LP) and the response to injury (IA/VA). However, there was no significant relation between the final lumen size (perimeter or area) and neointima formation (IA or MIT). This indicates that the final outcome of balloon injury is due to neointima formation (smooth muscle cell proliferation and deposition of matrix) and to other processes, such as vessel remodeling (smooth muscle cell migration and contraction of matrix).
Mechanisms for Effects of Vitamins C and E
The effect of the vitamins may be due to modification of vascular
redox state, as shown by decreased circulating lipid oxidation
(significant decreases in LDL TBARS and conjugated diene formation). In
addition, we previously demonstrated in this same model that another
antioxidant, probucol, also had beneficial effects on the vascular
response to injury.24 The response to injury of all cell
types present in the vessel wall may be altered by changing redox
state.
The beneficial effect of vitamins on remodeling may be due to improved endothelial cell regrowth and/or function. In the pig coronary after balloon injury, the endothelium begins to regenerate within 4 days, and by 7 to 14 days it has completely recovered the denuded area. Because vessels were studied only at 14 days after injury in the present investigation, it is not possible to determine whether treatment altered the rate of endothelial cell regrowth. Changes in endothelial cell function are supported by findings that in hypercholesterolemia there is impaired endothelial-dependent relaxation associated with decreased production of the endothelial-derived vasodilator nitric oxide.18 This is associated with increased production of superoxide by the vessel, suggesting that the redox state of the vessel is responsible for the impaired endothelial function. The importance of the endothelium in vascular remodeling is apparent from several studies. During development, vessels grow as blood flows into them and disappear when blood is shunted away. Similarly, in adults when there is increased flow (eg, graft anastomosis), the downstream vessel enlarges. Recent data suggest that remodeling in response to flow is mediated by the endothelium. If one removes the endothelium and decreases flow, vessel size does not decrease.45 Conversely, if a graft is placed in a high-flow hemodynamic environment and then switched to a low-flow environment, a neointima develops.46 Thus, it seems likely that alterations in the vessel redox state may have had beneficial effects on endothelial cell function, especially production of nitric oxide.
In addition to the endothelium, changes in vessel redox state may have
important consequences on remodeling by altering the nature of the
matrix produced by smooth muscle cells and fibroblasts. In this manner,
the vitamins may work by preventing vessel wall contraction or
scarring.14 15 In human restenosis, as in the porcine
model, the internal elastic lamina ruptures. This results in a loss of
vessel integrity and enables the vessel to be dilated beyond its normal
limits. However, over the ensuing weeks to months, new collagen and
matrix deposition restore vessel integrity. This matrix has tensile
strength and contracts to decrease vessel diameter and hence wall
tension. Antioxidant vitamins may delay or modify matrix deposition,
thereby enabling a larger diameter to be achieved. The finding that
internal elastic lamina FL was greater in all treatment groups (Table 2) supports this concept. It is possible that alterations in matrix
composition and architecture could be detrimental at later times,
particularly if it leads to aneurysm formation. Because the present
study was limited to 2 weeks after injury, extrapolating the results to
longer times and to human restenosis will require studies that last up
to 6 months. Future studies will also be required to determine the
precise effects of vitamins C and E on matrix composition and on the
enzymes responsible for matrix repair.
Implications for Future Studies
The pig is an excellent animal model for study of coronary artery
responses to balloon injury.28 30 Nonetheless,
extrapolation of the present study to humans is complicated by
differences in age (weanling pigs were studied), lipid status
(cholesterol was 70 mg/dL), vitamin metabolism, and initial vessel
pathology. For example, the changes in vitamin E levels achieved with
supplements were greater than those observed in humans. Baseline
vitamin E was 60 µg/mL and increased to 300 mg/mL. In healthy
humans (average age, 36 years), baseline vitamin E is 10 mg/mL and
increases to 20.3 mg/mL after 800 U/d for 8 weeks.47
Vitamin C is an essential vitamin in humans, while pigs can synthesize
vitamin C from dietary sources.40 Despite the fact that
steady-state circulating levels of vitamin C did not change in the
pigs, there was a significant effect of vitamin C supplementation on
lipid oxidation (Figs 5 and 6). This suggests that peak vitamin C
levels may have exerted a protective effect immediately after meals or
that storage and metabolism of vitamin C are different in lipids and
various tissues, as described previously.38 40 In
addition, the pig coronary studied here was initially a normal vessel
in contrast to the atherosclerotic human coronary that undergoes
angioplasty. The presence of inflammatory cells and oxidized lipids in
the atherosclerotic vessel makes the human coronary environment one in
which a state of oxidation is likely to exist. It is possible that
larger concentrations of antioxidants (or antioxidants effective
against specific mediators) may be necessary to alter the human
atherosclerotic vessel response to injury.
Finally, the present study strongly supports the concept
that vessel redox state is an important determinant of response to
injury. Superoxide production by vessels is dynamically regulated, as
shown by studies demonstrating 2-fold to 5-fold increases in response
to balloon injury16 and
hypercholesterolemia.18 In a subgroup of the injured LAD
vessels used for this study, there was a 2.3-fold increase in
superoxide production (data not shown). In response to treatment with
vitamins C and E together, there was a significant reduction in
superoxide production (60%) by the injured segment of the LAD
compared with the uninjured RCA or an adjacent uninjured segment of the
LAD (B.B. et al, unpublished data). These findings suggest that
treatment with vitamins C and E modified the production of superoxide
after injury. The hypothesis that excess active oxygen species
contribute to atherosclerosis and restenosis is supported by many
studies.19 Several antioxidants have been shown to limit
lipid accumulation in vessels of animals fed high-cholesterol
diets.20 21 48 49 Our recent study with the antioxidant
probucol in the pig coronary model showed that this antioxidant limited
neointima growth after balloon injury.24 The
present study supports this work and extends it by showing that
there is a correlation between response to injury and circulating LDL
oxidation state. In particular, because the greatest morphological
benefit occurred in group C + E, which also exhibited the greatest
change in lipid redox state, a cause-and-effect relationship is
possible. Future studies are required to determine which intracellular
mechanisms are the sources for oxygen radical production in injured
vessels and which active oxygen species
(O2-, OH-, and
H2O2) are most important. This information
should enable targeting of the vessel redox state with more specific
and powerful antioxidants.
|
Acknowledgments |
|---|
This work was supported in part by a grant from the Monsanto Corp. Dr Berk is an Established Investigator of the American Heart Association.
Received April 19, 1994; accepted October 4, 1994.
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References |
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O. Varenne, S. Pislaru, H. Gillijns, N. Van Pelt, R. D. Gerard, P. Zoldhelyi, F. Van de Werf, D. Collen, and S. P. Janssens Local Adenovirus-Mediated Transfer of Human Endothelial Nitric Oxide Synthase Reduces Luminal Narrowing After Coronary Angioplasty in Pigs Circulation, September 1, 1998; 98(9): 919 - 926. [Abstract] [Full Text] [PDF]
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M. Janiszewski, C. A Pasqualucci, L. C Souza, F. Pileggi, P. L da Luz, and F. R M. Laurindo Oxidized thiols markedly amplify the vascular response to balloon injury in rabbits through a redox active metal-dependent pathway Cardiovasc Res, August 1, 1998; 39(2): 327 - 338. [Abstract] [Full Text] [PDF]
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A. Lafont and D. Faxon Why do animal models of post-angioplasty restenosis sometimes poorly predict the outcome of clinical trials? Cardiovasc Res, July 1, 1998; 39(1): 50 - 59. [Full Text] [PDF]
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Z. S. Galis, K. Asanuma, D. Godin, and X. Meng N-Acetyl-Cysteine Decreases the Matrix-Degrading Capacity of Macrophage-Derived Foam Cells : New Target for Antioxidant Therapy? Circulation, June 23, 1998; 97(24): 2445 - 2453. [Abstract] [Full Text] [PDF]
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J. Rodes, G. Cote, J. Lesperance, M. G. Bourassa, S. Doucet, L. Bilodeau, O. F. Bertrand, F. Harel, R. Gallo, and J.-C. Tardif Prevention of Restenosis After Angioplasty in Small Coronary Arteries With Probucol Circulation, February 10, 1998; 97(5): 429 - 436. [Abstract] [Full Text] [PDF]
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G. L. Nunes, K. Robinson, A. Kalynych, S. B. King III, D. S. Sgoutas, and B. C. Berk Vitamins C and E Inhibit O2- Production in the Pig Coronary Artery Circulation, November 18, 1997; 96(10): 3593 - 3601. [Abstract] [Full Text]
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J. K. Miyashiro, V. Poppa, and B. C. Berk Flow-Induced Vascular Remodeling in the Rat Carotid Artery Diminishes With Age Circ. Res., September 19, 1997; 81(3): 311 - 319. [Abstract] [Full Text]
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 J.-C. Tardif, G. Cote, J. Lesperance, M. Bourassa, J. Lambert, S. Doucet, L. Bilodeau, S. Nattel, P. de Guise, and The Multivitamins and Probucol Study Group Probucol and Multivitamins in the Prevention of Restenosis after Coronary Angioplasty N. Engl. J. Med., August 7, 1997; 337(6): 365 - 372. [Abstract] [Full Text] [PDF]
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H. Rud Andersen, M. Mæng, M. Thorwest, and E. Falk Remodeling Rather Than Neointimal Formation Explains Luminal Narrowing After Deep Vessel Wall Injury : Insights From a Porcine Coronary (Re)stenosis Model Circulation, May 1, 1996; 93(9): 1716 - 1724. [Abstract] [Full Text]
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A. S. Baas and B. C. Berk Differential Activation of Mitogen-Activated Protein Kinases by H2O2 and O2- in Vascular Smooth Muscle Cells Circ. Res., July 1, 1995; 77(1): 29 - 36. [Abstract] [Full Text]
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