© 1997 American Heart Association, Inc.
Articles |
Consequences of Hyperhomocyst(e)inemia on Vascular Function in Atherosclerotic Monkeys
From the Veterans Affairs Medical Center (S.R.L., D.D.H.), and the Departments of Internal Medicine (S.R.L., D.J.P., F.M.F., D.D.H.) and Pharmacology (M.B.-T., F.M.F., D.D.H.), University of Iowa College of Medicine, Iowa City, Iowa; and Oregon Regional Primate Research Center, Beaverton, Ore (M.R.M.).
Correspondence to Steven R. Lentz, MD, PhD, Department of Internal Medicine, C303 GH, The University of Iowa, Iowa City, IA 52242. E-mail steven-lentz{at}uiowa.edu
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Abstract |
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Abstract Moderate elevation of plasma homocyst(e)ine is associated with increased risk for atherosclerotic vascular disease. In a previous study, we observed impaired vascular function in nonatherosclerotic monkeys with moderate hyperhomocyst(e)inemia. In this study, we tested the hypothesis that dietary intervention to lower plasma homocyst(e)ine corrects vascular dysfunction in atherosclerotic monkeys. Cynomolgus monkeys were fed an atherogenic diet that produces both hypercholesterolemia and moderate hyperhomocyst(e)inemia. After 17 months, the atherogenic diet was supplemented with B vitamins (5 mg folic acid, 400 µg vitamin B-12, and 20 mg vitamin B-6 daily) for 6 months. Total plasma homocyst(e)ine decreased from 12.8±2.8 to 3.5±0.3 µmol/L (n=9; mean±SE; P<.01) after vitamins were added to the diet, but plasma cholesterol remained elevated (522±63 versus 514±41 mg/dL; P>.05). In response to intra-arterial infusion of collagen, blood flow to the leg decreased by 30±3% and 38±5%, respectively, before and after vitamin supplementation (P>.05). In vivo responses of resistance vessels to endothelium-dependent vasodilators (acetylcholine or ADP) were impaired at baseline and did not improve after vitamin supplementation. In carotid artery studied ex vivo, relaxation to low doses of acetylcholine improved after vitamin supplementation, but maximal relaxation remained impaired. Ex vivo thrombomodulin anticoagulant activity was threefold higher in monkeys fed the atherogenic diet (with or without B vitamins) than in normal monkeys (P<.05). We conclude that normalization of plasma homocyst(e)ine is insufficient to restore normal vascular function in atherosclerotic monkeys with persistent hypercholesterolemia and that atherosclerosis, with or without hyperhomocyst(e)inemia, is associated with elevated thrombomodulin activity.
Key Words: atherosclerosis • endothelium • folate • homocysteine • thrombomodulin
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Introduction |
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An association between elevated plasma homocyst(e)ine concentration and atherosclerotic vascular disease has been observed in a large number of epidemiological studies.1 The term "homocyst(e)ine" is used to indicate that plasma homocysteine assays measure the total concentration of thiol, disulfide, and mixed disulfide adducts of homocysteine.2 Moderate hyperhomocyst(e)inemia, which may be caused by either genetic or dietary factors, occurs in up to 30% of patients with stroke, peripheral vascular disease, or myocardial infarction.1 3 Subclinical deficiencies of B folic acid, vitamin B-6, or vitamin B-12, which function in homocysteine metabolism, elevate plasma homocyst(e)ine to levels associated with vascular disease.4 5 6 7 8 Although supplementation of the diet with B vitamins is effective in lowering plasma homocyst(e)ine concentration,9 potential beneficial effects of vitamin supplementation on vascular function have not been evaluated in prospective studies.10
In a previous study, we observed impaired vasomotor regulation and altered endothelial antithrombotic function in monkeys with diet-induced moderate hyperhomocyst(e)inemia [plasma homocyst(e)ine >10 µmol/L].11 The magnitude of vascular dysfunction observed in hyperhomocyst(e)inemic monkeys was similar to that observed in atherosclerotic monkeys in several previous studies.12 13 14 More recently,15 we have found that monkeys fed atherogenic diet develop both hypercholesterolemia and moderate hyperhomocyst(e)inemia, a combination of risk factors that may be quite prevalent in humans with atherosclerosis.1 These observations suggested the possibility that hyperhomocyst(e)inemia may be one mechanism for impaired vascular function in atherosclerotic monkeys.
Vascular function normalizes within 1 month of decreasing plasma homocyst(e)ine concentration in nonatherosclerotic monkeys,11 and vascular function improves within 4 months when atherosclerotic monkeys are fed regression diet (which decreases plasma cholesterol and presumably also normalizes plasma homocyst(e)ine).16 We reasoned, therefore, that if hyperhomocyst(e)inemia is a major cause of vascular dysfunction in atherosclerotic monkeys, then normalization of plasma homocyst(e)ine through dietary supplementation with B vitamins should be sufficient to produce improvement in vascular function.
The goal of this study was to test the hypothesis that dietary intervention to decrease plasma homocyst(e)ine alters vascular function in atherosclerotic monkeys. Cynomolgus monkeys were fed atherogenic diet for 17 months to induce atherosclerotic lesions and then placed on atherogenic diet supplemented with B vitamins for 6 months. Plasma homocyst(e)ine concentration, vasomotor responses in vivo and ex vivo, carotid artery intimal and medial area, and thrombomodulin anticoagulant activity were measured before and after vitamin supplementation.
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Methods |
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Animals
Adult cynomolgus monkeys (Macaca fascicularis) were fed either normal diet (Purina Monkey Chow, Ralston Purina) or an atherogenic diet, which has been described in detail previously.17 18 The atherogenic diet contained 43% of total calories as fat, 0.7% cholesterol, and small amounts of B vitamins (approximately 1.0 µg vitamin B-12, 0.75 mg vitamin B-6, and <25 µg folic acid daily). Levels of plasma homocyst(e)ine in the atherosclerotic monkeys (14.6±1.7 µmol/L; n=24) were similar to or greater than levels previously found to be associated with vascular dysfunction in hyperhomocyst(e)inemic, nonatherosclerotic monkeys.11
A subset of monkeys (n=9) was fed atherogenic diet for 15 to 17 months and then fed atherogenic diet supplemented with B vitamins (5 mg folic acid, 400 µg cyanocobalamin, and 20 mg pyridoxine hydrochloride daily) for 5 to 6 months. Plasma homocyst(e)ine concentration, vasomotor responses in vivo and ex vivo, and thrombomodulin anticoagulant activity were measured in these monkeys before and after vitamin supplementation. The monkeys weighed 6.0±0.5 kg before vitamin supplementation and 6.1±0.5 kg after vitamin supplementation.
Experimental Protocol
Before receiving vitamin supplementation, animals were
sedated with ketamine hydrochloride (25 mg/kg IM) and
anesthetized with sodium pentobarbital (30 mg/kg IV). A
tracheotomy was performed, and animals were intubated and ventilated
with room air and supplemental oxygen. A nonobstructive multiple
sidehole catheter equipped with a Doppler transducer was inserted
into the right femoral artery and positioned in the distal aorta, and
the right femoral vein was cannulated for administration of
supplemental anesthesia (pentobarbital 15 mg/kg IV
as needed) and other drugs. Heart rate, respiration, and blood pressure
were monitored continuously.
Changes in blood flow to the leg were measured in response to intra-arterial infusion of collagen (150 mg/min for 10 minutes), and intra-arterial injection of serotonin (100 µg), acetylcholine (3x10-8, 1x10-7, and 3x10-7 mol), ADP (3x10-8, 1x10-7, and 3x10-7 mol), and sodium nitroprusside (1x10-8, 3x10-8, and 1x10-7 mol). Responses were monitored in vivo by quantitative angiography and Doppler measurement of hindlimb blood flow velocity. Cineangiograms of the distal descending aorta and the left iliac arterial tree were obtained as described previously.11 Quantitation of arterial lumen diameter was performed using computerized arterial lumen edge detection software19 as we have described previously.16 20 Velocity of blood flow to the leg was measured using the Doppler transducer at the time of angiography. By measuring velocity of flow (by Doppler) and aortic mean diameter (by angiography), blood flow to the leg was calculated.
At the end of the procedure, one common carotid artery was exposed, ligated proximally and distally with sutures, and the isolated segment of artery was removed and placed in oxygenated Krebs solution. Removal of one carotid artery did not produce stroke or other adverse effects in any monkeys and did not alter mean arterial pressure, which was 102±6 mm Hg before vitamin supplementation and 103±5 mm Hg after vitamin supplementation.
After receiving diet supplemented with B vitamins, animals again were anesthetized, and measurements of vasomotor responses in vivo to collagen, serotonin, acetylcholine, ADP, and nitroprusside were repeated. The remaining common carotid artery and a segment of thoracic aorta were removed and placed in oxygenated Krebs solution. Animals then were killed by administration of sodium pentobarbital (200 mg/kg IV) followed by exsanguination while under deep anesthesia. The protocol was approved by the University of Iowa Animal Care and Use Committee.
Vasomotor Responses in Carotid Artery
After removal of loose connective tissue, the common carotid
artery was cut into multiple 5-mm rings. Carotid artery rings were
suspended in an organ chamber containing oxygenated Krebs
buffer maintained at 37°C and connected to a force transducer to
measure changes in isometric tension. Rings were precontracted to a
tension of 2.0 g by stepwise addition of prostaglandin
F2
(1 to 3 µmol/L), and
relaxation dose-response curves were generated by cumulative
addition of acetylcholine (10-9 to
10-5 mol/L) or sodium nitroprusside
(10-9 to 10-5
mol/L).
Thrombomodulin Activity
Thrombomodulin-dependent protein C activation was measured
as described previously.11 After removal of adventitia and
loose connective tissue, segments of common carotid artery and thoracic
aorta were rinsed in Krebs solution and cut into multiple discs of
3 mm diameter (corresponding to an endothelial
surface area of approximately 7 mm2).
Arterial discs were incubated for 60 minutes at 37°C with
2.6 nmol/L human thrombin (Enzyme Research Laboratories) and
0.84 µmol/L human protein C (a generous gift of Dr Hans
Peter Schwarz, Immuno AG, Vienna). The reaction was stopped by addition
of a mixture of 25 µg/mL antithrombin III and 25 U/mL heparin,
and the amidolytic activity of activated protein C was measured
spectrophotometrically. One unit of activity was defined as the amount
of activated protein C generated in the presence of 0.75
ng/mL rabbit thrombomodulin (Kabi Pharmacia Hepar, Inc).
Histology and Morphometric Analysis
Sections of carotid artery were fixed in formalin,
embedded in paraffin, and stained with hematoxylin and eosin or
Verhoeff–van Gieson's stain. Intimal, medial, and luminal areas were
measured as described previously.17 Luminal area was
corrected for absence of pressure by measuring the length of the
internal elastic lamina.
Other Assays
Frozen plasmas were coded and air shipped under dry ice to Dr
Malinow's laboratory in Beaverton, Ore. Fasting plasma homocyst(e)ine
concentration was measured by high-performance liquid
chromatography and electrochemical detection, based on
the method of Smolin and Schneider,21 as described
previously.22 23 Plasma folate and vitamin B-12 were
measured by radioimmunoassay (Bio-Rad Quantaphase II, Bio-Rad
Diagnostics), and vitamin B-6 was measured by
radioenzymatic assay of pyridoxal 5'phosphate (Buhlman Laboratories
AG). Total plasma cholesterol was measured using methods
established by the Lipid Research Centers and standardized by the
Centers for Disease Control as described previously.24
Statistical Analysis
Statistical comparisons were performed using the paired,
two-tailed Student's t test to compare measurements in
monkeys before and after vitamin supplementation. A value of
P<.05 was used to define statistical significance.
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Results |
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Effects of Vitamin Supplementation on Plasma Homocyst(e)ine and Cholesterol
Adult cynomolgus monkeys were fed atherogenic diet for 16.5±0.4 months (mean±SE; n=9), and then fed atherogenic diet supplemented with B vitamins for 5.6±0.1 months. Before vitamin supplementation, these monkeys had moderately elevated levels of plasma homocyst(e)ine (12.8±2.8 µmol/L) and total plasma cholesterol (522±63 mg/dL). After the same monkeys were fed atherogenic diet supplemented with B vitamins for 5 to 6 months, plasma homocyst(e)ine concentration decreased to 3.5±0.3 µmol/L (n=9; P<.01) but plasma cholesterol remained elevated (P>.05) (Fig 1
).
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Vasomotor Responses in the Leg
Intra-arterial infusion of collagen, which
activates platelets in vivo,20 decreased
hindlimb blood flow by 30±3% before vitamin supplementation and by
38±5% after vitamins were added to the atherogenic diet (n=8;
P>.05). Platelet count in venous blood from the leg
decreased by similar amounts after infusion of collagen before and
after vitamin supplementation (38±5% and 37±3%, respectively).
Intra-arterial administration of serotonin
decreased hindlimb blood flow by 7±3% before vitamin supplementation
and by 15±4% after vitamin supplementation (n=9;
P>.05).
Compared with measurements obtained before supplementation of the
atherogenic diet, monkeys fed atherogenic diet supplemented with B
vitamins exhibited smaller increases in blood flow to the leg in
response to the highest doses of acetylcholine (n=9; P<.05)
(Fig 2A). Increases in blood flow in
response to ADP also tended to be smaller after vitamin
supplementation, although the differences did not achieve statistical
significance (Fig 2B). No differences in blood flow to the leg in
response to nitroprusside were observed after vitamin supplementation
(Fig 2C).
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Vasomotor Responses in Carotid Artery
Acetylcholine and nitroprusside each produced dose-dependent
relaxation of rings of common carotid artery (Fig 3). Relaxation of the carotid artery in
response to low doses of acetylcholine improved modestly after vitamin
supplementation, but responses to high doses were similar before and
after vitamin supplementation (Fig 3A). The highest dose of
acetylcholine (1x10-5 mol/L) relaxed
carotid artery rings by 53±9% before vitamin supplementation and by
55±9% after vitamin supplementation. Carotid arteries also were
slightly more responsive to low doses of nitroprusside after vitamin
supplementation (P<.05) (Fig 3B).
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Thrombomodulin Activity
Thrombomodulin-dependent protein C activation was measured
ex vivo in discs of common carotid artery before vitamin
supplementation and in both common carotid artery and thoracic aorta
after vitamin supplementation. Before vitamins were added to the diet,
thrombomodulin activity in carotid artery was approximately threefold
higher in atherosclerotic monkeys than in monkeys fed normal diet
(P<.05) (Fig 4). After
vitamin supplementation, carotid artery thrombomodulin activity
remained elevated, and thrombomodulin activity in thoracic aorta also
was increased in atherosclerotic compared with nonatherosclerotic
monkeys (Fig 4). In both carotid artery and aorta, thrombomodulin
activity decreased by >65% after in vitro removal of
endothelium, which indicates that most of the activity
measured was from thrombomodulin expressed on luminal
endothelium.
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Morphometry of Carotid Artery
Before vitamin supplementation, sections of common carotid artery
exhibited modest intimal thickening (Table 1). After atherogenic diet was
supplemented with B vitamins for 6 months, intimal thickening
progressed, with an approximate doubling of intimal area
(P<.05). Despite a significant increase in intimal area,
luminal area did not decrease after vitamin supplementation, which
indicates that vascular remodeling occurred.18 25
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Discussion |
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One major finding of this study is that atherosclerosis, with or without hyperhomocyst(e)inemia, is associated with elevated thrombomodulin activity in monkeys. Another major finding is that supplementation of atherogenic diet with B vitamins decreases plasma homocyst(e)ine concentration, but does not correct vascular dysfunction, in atherosclerotic monkeys. This finding is consistent with previous observations that oral administration of B vitamins decreases plasma homocyst(e)ine concentration in humans.9 On the basis of such observations, it has been suggested that interventions directed toward lowering plasma homocyst(e)ine may produce clinical benefit in humans with atherosclerotic vascular disease.10 26 Our results, however, indicate that dietary intervention to correct hyperhomocyst(e)inemia is insufficient to normalize vascular function or prevent progression of structural lesions in monkeys with persistent hypercholesterolemia.
We chose to study the effect of B vitamins on vascular function in a cohort of monkeys that had been fed atherogenic diet for 15 to 17 months. These monkeys had coexisting hypercholesterolemia and moderate hyperhomocyst(e)inemia, a combination of factors that may be common in humans with vascular disease.1 Before vitamin supplementation, the monkeys had structural lesions in the carotid artery that were characteristic of early atherosclerosis, with modest intimal thickening, and moderately severe vascular dysfunction. As we have observed previously,27 28 these animals had impaired endothelium-dependent responses in both large and small vessels and augmented vasoconstrictor responses to intra-arterial infusion of collagen, which is a potent activator of platelets in vivo.20
After the monkeys were fed atherogenic diet supplemented with B vitamins for 6 months, the structural lesions in the carotid artery progressed, with evidence of vascular remodeling.18 25 Vascular function remained moderately impaired after the diet was supplemented with B vitamins. These findings indicate that correction of hyperhomocyst(e)inemia, without concomitant correction of hypercholesterolemia, did not prevent progression of structural lesions or restore normal vascular function in atherosclerotic monkeys. These results, however, do not exclude the possibility that decreased plasma homocyst(e)ine concentration may have protected the carotid artery from progression of dysfunction. Definitive conclusions regarding potential protective effects of B vitamins on vascular function during progression of atherosclerosis will require a concurrently studied comparison group fed atherogenic diet without vitamins. It also is possible that vitamin supplementation may have greater protective effects on vascular function in animals with higher initial levels of plasma homocyst(e)ine or lower initial levels of plasma cholesterol.
In addition to its effects on endothelial
vasomotor function, hyperhomocyst(e)inemia appears to alter
endothelial antithrombotic function. Homocysteine
inhibits the protein C anticoagulant pathway in vitro by inactivating
thrombomodulin, an essential endothelial
cofactor.29 30 We have observed impaired thrombomodulin
activity in large vessels of hyperhomocyst(e)inemic
monkeys.11 In this study, thrombomodulin activity in
atherosclerotic common carotid artery and thoracic aorta was
approximately threefold higher than in monkeys fed normal diet (Fig 4
)
and approximately fourfold higher than in nonatherosclerotic,
hyperhomocyst(e)inemic monkeys.11 Thrombomodulin activity
in carotid artery did not change significantly after the atherogenic
diet was supplemented with B vitamins. These findings suggest that
thrombomodulin may be upregulated in atherosclerotic vessels, possibly
as a compensatory antithrombotic response to
endothelial injury. This interpretation is
consistent with the detection of increased amounts of
thrombomodulin in the neointima of rabbit aorta after
balloon injury31 and the observation that plasma levels of
thrombomodulin are elevated in patients with peripheral or
coronary artery disease.32
The atherosclerotic monkeys in this study had impaired carotid
artery responses to nitroprusside, an
endothelium-independent nitrovasodilator. We have
observed impairment of vasodilator responses to nitroprusside in
previous studies of large vessels from atherosclerotic or
hyperhomocyst(e)inemic monkeys.11 28 The observation that
carotid artery responses to low doses of nitroprusside, and, to a
lesser extent, acetylcholine, improved after vitamin supplementation
(Fig 3) suggests that hyperhomocyst(e)inemia may inactivate
nitric oxide derived from both exogenous and endogenous
sources, possibly through oxidative mechanisms.33 However,
these data do not allow us to determine whether decreased
responsiveness to nitrovasodilators was caused by inactivation of
nitric oxide or by impairment of relaxation of vascular smooth muscle.
To address this question, it would be useful to examine effects of
hyperhomocyst(e)inemia on relaxation that is not mediated by nitric
oxide or cGMP.
Dietary deficiency of folic acid, vitamin B-6, or vitamin B-12,
which are essential for normal homocysteine metabolism, is
associated with moderate hyperhomocyst(e)inemia in
humans.4 5 7 Compared with monkeys fed normal diet,
monkeys fed unsupplemented atherogenic diet had lower plasma levels of
each of these B vitamins (Table 2).
Plasma levels of folic acid, vitamin B-6, and vitamin B-12 increased
after addition of these vitamins to the diet (Table 2). Therefore, the likely cause of
hyperhomocyst(e)inemia in the atherosclerotic monkeys was dietary
deficiency of B vitamins, rather than altered vitamin bioavailability
induced by atherogenic diet.
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Thus, although it is established that oral administration of B vitamins is effective in lowering plasma homocyst(e)ine concentration, the potential beneficial effects of vitamin supplementation on vascular function and prevention of complications of atherosclerotic vascular disease remain to be tested. It is likely that interventions directed at lowering both cholesterol and homocyst(e)ine may be necessary to effectively correct vascular dysfunction and prevent progression of atherosclerosis.
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Acknowledgments |
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This work was supported by the Office of Research and Development, Department of Veterans Affairs, National Institutes of Health grants HL-14388, NS-24621, HL-16066, AG-10269, DK-25295, and RR-00163, and the Roy J. Carver Charitable Trust. We thank Robert M. Brooks, II, for technical assistance with carotid artery vasomotor studies and Pam Tompkins for technical assistance with morphometric measurements.
Received September 25, 1996; accepted March 27, 1997.
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J. Zhou, J. Moller, C. C. Danielsen, J. Bentzon, H. B. Ravn, R. C. Austin, and E. Falk Dietary Supplementation With Methionine and Homocysteine Promotes Early Atherosclerosis but Not Plaque Rupture in ApoE-Deficient Mice Arterioscler. Thromb. Vasc. Biol., September 1, 2001; 21(9): 1470 - 1476. [Abstract] [Full Text] [PDF]
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S. R. Lentz, D. J. Piegors, M. R. Malinow, and D. D. Heistad Supplementation of Atherogenic Diet With B Vitamins Does Not Prevent Atherosclerosis or Vascular Dysfunction in Monkeys Circulation, February 20, 2001; 103(7): 1006 - 1011. [Abstract] [Full Text] [PDF]
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Z. Bagi, Z. Ungvari, L. Szollar, and A. Koller Flow-Induced Constriction in Arterioles of Hyperhomocysteinemic Rats Is Due to Impaired Nitric Oxide and Enhanced Thromboxane A2 Mediation Arterioscler. Thromb. Vasc. Biol., February 1, 2001; 21(2): 233 - 237. [Abstract] [Full Text] [PDF]
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J. C. Chambers, P. M. Ueland, O. A. Obeid, J. Wrigley, H. Refsum, and J. S. Kooner Improved Vascular Endothelial Function After Oral B Vitamins : An Effect Mediated Through Reduced Concentrations of Free Plasma Homocysteine Circulation, November 14, 2000; 102(20): 2479 - 2483. [Abstract] [Full Text] [PDF]
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R. H. Boger, S. M. Bode-Boger, K. Sydow, D. D. Heistad, and S. R. Lentz Plasma Concentration of Asymmetric Dimethylarginine, an Endogenous Inhibitor of Nitric Oxide Synthase, Is Elevated in Monkeys With Hyperhomocyst(e)inemia or Hypercholesterolemia Arterioscler. Thromb. Vasc. Biol., June 1, 2000; 20(6): 1557 - 1564. [Abstract] [Full Text] [PDF]
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Z. Ungvari, E. Sarkadi-Nagy, Z. Bagi, L. Szollar, and A. Koller Simultaneously Increased TxA2 Activity in Isolated Arterioles and Platelets of Rats With Hyperhomocysteinemia Arterioscler. Thromb. Vasc. Biol., May 1, 2000; 20(5): 1203 - 1208. [Abstract] [Full Text] [PDF]
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Z. Ungvari, P. Pacher, K. Rischak, L. Szollar, and A. Koller Dysfunction of Nitric Oxide Mediation in Isolated Rat Arterioles With Methionine Diet–Induced Hyperhomocysteinemia Arterioscler. Thromb. Vasc. Biol., August 1, 1999; 19(8): 1899 - 1904. [Abstract] [Full Text] [PDF]
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S. J. Kittner, W. H. Giles, R. F. Macko, J. R. Hebel, M. A. Wozniak, R. J. Wityk, P. D. Stolley, B. J. Stern, M. A. Sloan, R. Sherwin, et al. Homocyst(e)ine and Risk of Cerebral Infarction in a Biracial Population : The Stroke Prevention in Young Women Study Stroke, August 1, 1999; 30(8): 1554 - 1560. [Abstract] [Full Text] [PDF]
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S. R. Lentz, J. A. Fernandez, J. H. Griffin, D. J. Piegors, R. A. Erger, M. R. Malinow, and D. D. Heistad Impaired Anticoagulant Response to Infusion of Thrombin in Atherosclerotic Monkeys Associated With Acquired Defects in the Protein C System Arterioscler. Thromb. Vasc. Biol., July 1, 1999; 19(7): 1744 - 1750. [Abstract] [Full Text] [PDF]
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L. H. Kuller and R. W. Evans Homocysteine, Vitamins, and Cardiovascular Disease Circulation, July 21, 1998; 98(3): 196 - 199. [Full Text] [PDF]
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C. A. Hathaway, D. D. Heistad, D. J. Piegors, and F. J. Miller Jr Regression of Atherosclerosis in Monkeys Reduces Vascular Superoxide Levels Circ. Res., February 22, 2002; 90(3): 277 - 283. [Abstract] [Full Text] [PDF]
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