© 1997 American Heart Association, Inc.
Articles |
ß-VLDL Hypercholesterolemia Relative to LDL Hypercholesterolemia Is Associated With Higher Levels of Oxidized Lipoproteins and a More Rapid Progression of Coronary Atherosclerosis in Rabbits
From the Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, O & N, Belgium.
Correspondence to Paul Holvoet, PhD, Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, O & N, Herestraat 49, B-3000 Leuven, Belgium. E-mail paul.holvoet{at}med.kuleuven.ac.be
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Abstract |
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Abstract The accumulation of the oxidized apolipoprotein, apoB-100, containing lipoproteins in the arterial wall and the progression of coronary atherosclerotic lesions in rabbits with ß-VLDL and LDL hypercholesterolemia was compared. In New Zealand White (NZW) rabbits on a 0.125% cholesterol diet, LDL cholesterol levels increased from 14±1 mg/dL (mean±SEM; n=9) to 170±34 mg/dL (n=10, P=.0002). On 0.5% cholesterol, LDL cholesterol levels were similar, but ß-VLDL cholesterol levels increased from 60±4 mg/dL (n=10) to 550±75 mg/dL (n=8; P<.0001). In Watanabe heritable hyperlipidemic (WHHL) rabbits, LDL cholesterol levels were 2.3 -fold higher (n=13; P<.0001) than in NZW rabbits on 0.5% cholesterol, whereas their ß-VLDL cholesterol levels were 3.7-fold lower (P<.0001), resulting in similar total cholesterol levels. At 2 months, mean intimal areas of lesions in the coronary arteries of NZW rabbits on 0.125% cholesterol were 0.13±0.045 mm2 (n=4; mean±SEM) and were 5.8-fold (n=4; P=.016) and 2.0-fold (n=6; P=NS versus 0.125% cholesterol and P=.014 versus 0.5% cholesterol) higher in NZW rabbits on 0.5% cholesterol and in WHHL rabbits, respectively. At 5 months, mean intimal areas were 0.47±0.088 mm2 (n=6) in NZW rabbits on 0.125% cholesterol and were 4.5-fold (n=4; P=.0001) and 2.0-fold (n=7; P=.012 and P=.0019) higher in rabbits on 0.5% cholesterol and in WHHL rabbits, respectively. Levels of oxidized apoB-100 containing lipoproteins (both ß-VLDL and LDL) in the lesions correlated with mean intimal area (r=.88; n=31; P<.0001) of those lesions and with the plasma levels of total ß-VLDL/LDL (r=.72; P<.0001). Levels of oxidized apoB-100 containing lipoproteins in the arterial wall correlate with progression of hypercholesterolemia- induced coronary atherosclerotic lesions. Plasma levels of ß-VLDL relative to similar increases in LDL result in a more pronounced accumulation of oxidized apoB-100 containing lipoproteins in the arterial wall and in the plasma and a more rapid progression of coronary atherosclerosis.
Key Words: coronary atherosclerosis • oxidized ß-VLDL • oxidized LDL.
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Introduction |
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Elevated serum LDL cholesterol levels are an important factor in atherogenesis.1–3 The finding that premature atherosclerosis develops in animals and humans with deficiencies of functional LDL receptors has suggested alternative pathways for LDL uptake. When exposed to Cu2+, LDL undergoes oxidative modification when incubated in vitro with endothelial cells, smooth muscle cells, or macrophages,4,5 and oxidized LDL is rapidly taken up by monocytes/macrophages via scavenger receptors, transforming them into foam cells, which are essential components of fatty streaks and fibrofatty plaques. Accumulation of foam cells in the subendothelial space constitutes an early event in atherosclerosis.4–6 A deletion of four amino acids from the cysteine-rich ligand binding domain of the LDL receptor7,8 preventing the LDL receptor-mediated uptake of LDL particles in the liver results in a dramatic increase of LDL cholesterol that is associated with accelerated atherosclerosis. Probucol was found to reduce the rate of development of fatty streak lesions in the aorta of Watanabe heritable hyperlipidemic (WHHL) rabbits, suggesting that limiting oxidative LDL modification may prevent foam cell transformation of macrophages and progression of atherosclerosis.9,10 Gene therapy of WHHL rabbits with an adenoviral vector containing the rabbit LDL receptor cDNA resulted in a 3.3-fold decrease of LDL cholesterol, suggesting that these rabbits are suitable for studying the role of LDL and oxidized LDL in the progression of atherosclerotic lesions.11
In cholesterol-fed animals, a large fraction of the plasma cholesterol present in lipoproteins are of very low density (VLDL), have ß-electrophoretic mobility (ß-VLDL) and are taken up via the LDL receptor.12–14 Hornick et al15 have demonstrated that the VLDLs of rabbits contain apoB-100 and not apoB-48 and that apoB-100 is secreted from the liver virtually exclusively as VLDLs, which are converted to LDL by the cholesteryl ester transfer protein (CETP). Therefore, cholesterol-fed rabbits are suitable to study the role of ß-VLDL and oxidized ß-VLDL in the progression of atherosclerosis.
In the present study, a monoclonal antibody specific for oxidatively modified apoB-10016 was used to measure levels of oxidized apoB-100 containing lipoproteins (ß-VLDL and LDL), in atherosclerotic lesions and in plasma. The aim of the study was to investigate the correlation between levels of oxidized ß-VLDL and LDL and the progression of lesions in coronary arteries of hypercholesterolemic rabbits that are primarily due to smooth muscle cell proliferation and foam cell formation and not to monocyte/macrophage accumulation.
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Methods |
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Animals
Three-month-old New Zealand White rabbits (3.0±0.12 kg body weight; mean±SEM of 66 rabbits) were housed in a room controlled to 18±1°C temperature, 50±2% humidity, on a 12 hour light/dark cycle. Atherosclerosis was induced by feeding the animals a 0.125% or a 0.5% cholesterol-rich diet at a daily amount of 150g. The atherogenic diet was prepared by spraying normal rabbit chow (Hope Farms) with cholesterol dissolved in ethyl-ether and allowing the solvent to evaporate in a fume hood. Water was provided ad libitum. All experimental procedures in these animals were performed in accordance with protocols approved by the Institutional Animal Care and Research Advisory Committee. Male, homozygous New Zealand White WHHL rabbits, obtained from Charles River (Cléon, France), were crossed with female, NZW rabbits and heterozygous female offspring was backcrossed with the homozygous male.
Blood Sampling
Venous blood samples from the rabbits were collected on 0.1 vol
of 0.1 mol/L citrate, containing 1 mmol/L EDTA, 20
µM vitamin E, 10 µM butylated hydroxytoluene, 20 µM
dipyridamole and 15 mmol/L theophylline to
prevent in vitro LDL oxidation and platelet activation. Blood
samples were centrifuged at 3000g for 15 minutes at
room temperature within 1 hour of collection and stored at -20°C
until the assays were performed.
Histomorphometric and Immunohistochemical Analyses
The left main coronary arteries were dissected free of
the heart; arterial specimens were submerged within 30
minutes after removal in PBS (pH 7.4), containing 4% sucrose, 20 µM
vitamin E, 10 µM butylated hydroxytoluene as antioxidants, and 1
mmol/L EDTA. Specimens were then snap-frozen in liquid nitrogen
and stored at -80°C. Frozen 7 µm sections from the proximal
0.5 to 0.7 mm segment of the artery were stained with hematoxylin
and eosin and with oil red 0 or immunostained as described
below. Six to eight sections at a distance of 84 µm were
analyzed for each rabbit and mean values were calculated. The
length of the analyzed segment thus ranged between 0.5 and
0.7 mm. Morphometric parameters of atherosclerotic
lesions were measured by planimetry using a Leica 2 Quantimet color
image analyzer. The area within the external elastic lamina,
the internal elastic lamina, and the lumen were measured. "Media"
was defined as the area between the internal and external elastic
lamina. "Intima" was defined as the area within the internal
elastic lamina not occupied by vessel lumen. The percentage of
stenosis was calculated as the ratio between intima and the
total area within the internal elastic lamina, multiplied by 100.
Maximal stenosis percentage was defined as the maximal value of
all stenosis percentage values in one artery. Atherosclerotic
lesions were classified as described previously.
17
Oxidized apoB-100 containing lipoproteins were detected with the specific monoclonal antibody mAb-4E6,16 alkaline-phosphatase conjugated rabbit-anti-mouse IgG antibodies, and the fuchsin alkaline phosphatase substrate system (Dako) and the mean intensities/mm2 were measured in the color image analyzer. Specificity of immunostaining was confirmed by inhibition of staining with excess of copper-oxidized LDL, but not with native LDL or with malondialdehyde-modified albumin. The staining co-localized with that of monoclonal antibody mAb-13F6, specific for apoB-100.
Immunostaining of smooth muscle cells and
monocytes/macrophages was performed with a cross-reacting
murine monoclonal antibody against human 
-actin (clone 1A4; Sigma)
or a cross-reacting rat monoclonal antibody against the common
leukocyte antigen/CD45 (clone 30F11.1; Pharmingen).
Endothelial cells were immunostained with
rabbit anti-von Willebrand Factor antibodies (Dakopatts).
Proliferating cells were immunostained with the monoclonal
mouse anti-proliferating cell nuclear antigen (clone PC10;
Dakopatts).
Quantitation of Oxidized apoB-100 Containing Lipoproteins in
Plasma
Plasma levels of oxidized apoB-100 containing lipoproteins,
ß-VLDL and LDL, were determined in a mAb-4E6 based ELISA, as
described elsewhere.16 The lower limit of
sensitivity of the ELISA is 0.020 mg/dL for human
copper-oxidized and human malondialdehyde-modified LDL and 20
mg/dL for native LDL. Intra- and inter-assay coefficients of
variation are 10 and 12%, respectively. When copper-oxidized LDL
was added to human plasma at a final concentration of 0.25 and 2
mg/dL, respectively, recoveries were 90 and 95%,
respectively.16
Measurement of Cholesterol Levels
Plasma total cholesterol levels were determined
using a standard enzymatic colorimetric assay
(Boehringer Mannheim). To investigate alterations in
cholesterol levels of different lipoprotein components,
ß-VLDL, LDL and HDL particles were separated by fast peptide liquid
chromatography.18 Rabbit plasma
in the amount of 200 µL was applied onto a Superose 6 column and a
Superdex 200HR column both of which were serially connected and then
eluted at 0.5 mL/min with PBS containing 1 mg/mL EDTA
(P=7.5).11 Fractions were collected (1
minute, 0.5 mL), and cholesterol levels were determined
using the enzymatic colorimetric assay.
Lipoprotein: Preparation and Modification
Lipoproteins isolated by gelfiltration were sterilized by
filtration, using a 0.45 µm low protein binding filter (Millex,
Millipore Corp) and stored at 4°C under nitrogen. Copper-oxidized
ß-VLDL, LDL, and HDL were prepared by incubation with copper chloride
(final concentration 640 µM for 16 hours) as described
elsewhere19 and the extent of lysine substitution
was determined by measurement of thiobarbituric acid reactive
substances.20
Statistical Analysis
All data were expressed as mean±SEM. Significance of difference
in plasma lipid values, size of atherosclerotic lesions, and levels of
oxidized ß-VLDL and LDL in plasma and in atherosclerotic lesions was
determined by Student's t test. Values of P<.05
were considered statistically significant. Correlation coefficients
were calculated according to Spearman using logarithmically transformed
data.
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Results |
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Cholesterol Levels in Plasma and Separated Lipoprotein Fractions
Total plasma cholesterol levels were determined by enzymatic assay. ß-VLDL, LDL, and HDL fractions were separated by gelfiltration and cholesterol levels were measured in isolated fractions. The lipoprotein distribution profiles in control NZW rabbits, NZW rabbits on 0.125 or 0.5% cholesterol, and in WHHL rabbits are illustrated in Fig 1
.
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), NZW rabbit on 0.125%
cholesterol (
), an NZW rabbit on 0.5%
cholesterol (
), and a WHHL rabbit (
).
Lipoprotein fractions were separated by fast peptide liquid
chromatography, and cholesterol levels were
determined by enzymatic assay. Data represent mean data of 3
rabbits per group.
Prediet baseline values in NZW rabbits (age 12 weeks at the start of
the study were 57±5 mg/dL (mean±SEM, n=9) for total
cholesterol, 7.0±0.7 mg/dL for ß-VLDL
cholesterol, 14±1 mg/dL for LDL
cholesterol (Fig 2). Feeding
of 0.125% (wt/wt) cholesterol resulted in a 3.8-fold
increase of total cholesterol due to a 9-fold increase in
ß-VLDL and a 12-fold increase in LDL cholesterol (Fig 2).
Feeding of 0.5% cholesterol resulted in a 14-fold increase
of total cholesterol. LDL cholesterol levels
were similar to those in rabbits on 0.125% cholesterol,
but VLDL cholesterol levels were 9-fold higher (Fig 2). The
cholesterol-rich diets did not affect body weight, number
of white or red blood cells, number of blood platelets, or
hemoglobulin content (data not shown). Whereas total
cholesterol levels in WHHL rabbits were similar to those in
NZW rabbits on 0.5% cholesterol, their lipoprotein
distribution profiles were significantly different. The increase in
total cholesterol in WHHL rabbits was primarily due to an
increase in LDL cholesterol (3.4-fold higher than in NZW
rabbits on 0.5% cholesterol), and to a lesser extent due
to an increase of ß-VLDL cholesterol (3.7-fold lower)
(Fig 2). HDL cholesterol levels in those rabbits were
34±2.4 mg/dL, 30±2.7 mg/dL, 16±2.0 mg/dL and
14±1.3 mg/dL, respectively.
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), NZW
rabbits on 0.125% cholesterol (
), NZW rabbits on 0.5%
cholesterol (
), and WHHL rabbits (
ß-VLDL, LDL, and HDL isolated by gelfiltration were oxidized in vitro
by incubation with copper chloride. Fig 3
shows that oxidized rabbit ß-VLDL and LDL and oxidized human LDL
inhibited the binding of mAb-4E6 to immobilized human
oxidized LDL to a similar extent. Because the oxidized rabbit ß-VLDL
and LDL showed a very similar interaction with mAb-4E6, a mAb-4E6-based
ELISA16 was used to measure the levels of
oxidized apoB-100 containing lipoproteins (sum of ß-VLDL and LDL) in
freshly frozen plasma samples of cholesterol-fed NZW
rabbits and WHHL rabbits. Plasma levels of oxidized apoB-100 containing
lipoproteins were 0.22±0.022 mg/dL in control NZW rabbits on
normal chow; they were 3-fold higher in NZW rabbits on 0.125%
cholesterol, 8-fold higher in NZW rabbits on 0.5%
cholesterol, and 6-fold higher in WHHL rabbits (Fig 2
).
Storage of the samples at -80°C for up to 6 months and up to 4
freezing and thawing cycles did not result in an increase of these
levels, suggesting that the addition of the antioxidants and of the
antiplatelet drugs efficiently prevented the in vitro oxidation of
LDL.
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Plasma levels of oxidized apoB-100 containing lipoproteins in the plasma of control NZW rabbits were too low to measure their distribution in the different lipoprotein fractions. Oxidized ß-VLDL levels were not detectable in NZW rabbits on 0.125% cholesterol, 1.28±0.12 mg/dL in NZW rabbits on 0.5% cholesterol, and 0.13±0.009 mg/dL (P<.0001 versus 0.5% cholesterol) in WHHL rabbits. Oxidized LDL levels were 0.49±0.056 mg/dL in NZW rabbits on 0.125% cholesterol, 0.52±0.021 mg/dL in NZW rabbits on 0.5% cholesterol (P=NS versus 0.125% cholesterol), and 1.09±0.073 mg/dL in WHHL rabbits (P<.0001 versus 0.125% cholesterol and versus 0.5% cholesterol). As expected, no immunoreactive material was detected in the HDL fractions. Plasma levels of oxidized ß-VLDL/LDL correlated with the plasma levels of total ß-VLDL/LDL (r=0.76; P<.0001).
Histomorphometric and Immunohistochemical Analysis of
Coronary Arteries
Feeding of normal chow for up to 5 months did not produce intima
formation in the left coronary arteries. Feeding of 0.125%
cholesterol induced neointima formation in the
coronary arteries with a mean intimal cross-sectional area of
0.13±0.045 mm2 (n=4) at 2 months and of
0.47±0.18 (n=6) mm2 at 5 months (Fig 4). The intima/media ratio increased to
0.14±0.070 and 0.41±0.17, respectively (Fig 4). The mean intimal area
of lesions in coronary arteries of rabbits on 0.5%
cholesterol was 5.8-fold higher at 2 months and 4.5-fold
higher at 5 months, whereas the intima/media ratios were, respectively,
5.2- and 5.4-fold higher than in rabbits on 0.125%
cholesterol (Fig 4). The mean intimal area of lesions in
coronary arteries in WHHL rabbits at 2 months was 2-fold higher
than in NZW rabbits on 0.125% cholesterol, but was
2.9-fold lower than in rabbits on 0.5% cholesterol with
corresponding values at 5 months that were 2-fold higher and 2.2-fold
lower, respectively (Fig 4).
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Fig 5 illustrates the accumulation of
oxidized apoB-100 containing lipoproteins (immunostained
red with the monoclonal antibody mAb-4E6) in lesions in the
coronary arteries of WHHL rabbits at 2 and 5 months. Fig 5 also
illustrates that lesions contained primarily smooth muscle cells
(immunostained red with the monoclonal antibody 1A4) and
not monocytes/macrophages (as evidenced by lack of
immunostaining of cells with the monoclonal antibody
30F11.1). The distribution of oxidized apoB-100 containing lipoproteins
in coronary lesions of cholesterol-fed NZW rabbits
was very similar (data not shown).
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The monoclonal antibody mAb-4E6 did not detect immunoreactive material
in the coronary arteries of rabbits fed a normal chow. Total
absorbance measured in the immunostained intima of left
coronary arteries of rabbits on 0.125% cholesterol
increased to 0.37±0.11 absorbance units at 2 months and to 0.87±0.18
at 5 months. The amounts of oxidized apoB-100 containing lipoproteins
in coronary arteries of rabbits on 0.5%
cholesterol were 4.0- and 2.5-fold higher than those in
rabbits on 0.125% cholesterol (Fig 4). The amounts in
coronary arteries of WHHL rabbits were 1.4- and 1.6- fold
higher than in rabbits on 0.125% cholesterol, but were
2.8- and 1.7-fold lower than in NZW rabbits on 0.5%
cholesterol (Fig 4).
Concentration-dependent decrease of mean intensities/mm2 in coronary artery sections of NZW rabbits on 0.5% cholesterol was obtained when in vitro oxidized LDL, ranging between 500 (90% inhibition) and 50 µg/mL (15% inhibition), was added to the antibody solution. Concentration-dependent decrease of mean intensities/mm2 in coronary artery sections of NZW rabbits on 0.125% cholesterol was obtained when in vitro oxidized LDL, ranging between 125 (90% inhibition) and 15 µg/mL (15% inhibition), was added to the antibody solution. Those data suggested that the absorbance was proportional to the amount of oxidized ß-VLDL/LDL in the lesions. The intra-assay variation coefficient was 8% (n=20), whereas the inter-assay variation coefficient was 16% (4 independent measurements of 10 different sections).
A positive correlation (n=31; 0.88; P<.0001) was observed
between the amount of oxidized ß-VLDL/LDL in the lesions and the
plasma levels of ß-VLDL/LDL (r=0.72; P<.0001) (data not
shown). A positive correlation also was observed between the amounts of
oxidized apoB-100 containing lipoproteins in the lesions and the mean
intimal area of the lesions (n=31; 0.88; P<.0001) in the
left main coronary arteries of NZW rabbits on 0.125%
cholesterol (n=10) or on 0.5% cholesterol
(n=8) and of WHHL rabbits (n=13) (Fig 6).
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Discussion |
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The present study demonstrates that hypercholesterolemia, either diet-induced in cholesterol-fed NZW rabbits or associated with LDL receptor deficiency in WHHL rabbits, is associated with progression of fibrofatty plaques in the coronary arteries. The increase of ß-VLDL cholesterol in NZW rabbits on 0.5% cholesterol induced a 2- to 3-fold more rapid progression of lesions than did the increase of LDL cholesterol in WHHL rabbits, although both groups had similar total cholesterol levels. Lesion amounts of oxidized apoB-100 containing lipoproteins, primarily ß-VLDL and LDL, correlated with the progression of the coronary lesions that were primarily due to smooth muscle cell proliferation and foam cell formation and not to monocyte/macrophage accumulation. Lesion amounts of oxidized apoB-100 containing lipoproteins were significantly higher in NZW rabbits on the high cholesterol diet than in WHHL rabbits, suggesting a more pronounced infiltration and oxidation of ß-VLDL than of LDL in the arterial wall. The significant correlation between plasma levels of ß-VLDL/LDL and the amounts of oxidized ß-VLDL/LDL in the lesions may indicate that (1) the oxidized ß-VLDL/LDL in the lesions are directly derived from the plasma oxidized ß-VLDL/LDL that infiltrate in the vessel wall, that (2) both the lesion amounts and the plasma levels of oxidized ß-VLDL/LDL reflect similar oxidative stresses both in the vessel wall and in the plasma of hypercholesterolemic rabbits, or that (3) there is backdiffusion of oxidized ß-VLDL/LDL generated in the vessel wall in the blood.
Previously, it has been demonstrated that damage or dysfunction of endothelium may reduce its effectiveness to act as a selectively permeable barrier to plasma components, including cholesterol-rich lipoprotein remnants. Lipid peroxidation can induce endothelial cell injury/dysfunction and endothelial cell injury by lipid hydroperoxides may increase the uptake of LDL into the vessel wall.21 Recently, we have demonstrated a positive correlation between the extent of endothelial injury and the oxidation of LDL in chronic renal failure patients.16
In the presence of hyperlipoproteinemia, endothelial hyperpermeability and accumulation of subendothelial matrix proteins may favor intimal uptake and retention of ß-VLDL and LDL. Accumulation of apoB-100-containing lipoproteins in the arterial wall following hypercholesterolemia induces alterations in sulfated glycosaminoglycans of matrix proteoglycans, resulting in enhanced retention of those lipoproteins.22 The reversible interaction of those lipoproteins with those glycosaminoglycans selects particles with a high affinity that are more prone to oxidation.23 Fractional rates of efflux of arterial ß-VLDL/LDL have been found to be decreased in lesion-susceptible areas, suggesting that the focal increases in ß-VLDL/LDL concentration observed in those sites are due to localized differences in ß-VLDL/LDL retention and rate of ß-VLDL/LDL degradation.24
Local oxidation of trapped ß-VLDL/LDL may generate lipid-derived inflammatory mediators, such as oxysterols, peroxidized fatty acids, and lysophospholipids that induce atherogenic monocytic inflammatory responses in arterial walls,25 resulting in the generation of macrophage foam cells and the initiation of fatty streaks.26 Oxidized LDL are more effective than oxidized ß-VLDL in recruiting monocytes and inducing macrophage foam cell generation in lesions in the thoracic and abdominal aorta of hypercholesterolemic rabbits.10,24,27 The present study, however, demonstrates that the progression of atherosclerotic lesions in the coronary arteries of hypercholesterolemic rabbits is primarily due to smooth muscle cell proliferation and transformation in foam cells and not to monocyte/macrophage recruitment and foam cell generation. The data suggest a correlation between the oxidation of ß-VLDL and LDL and smooth muscle cell proliferation and foam cell proliferation that is more pronounced in ß-VLDL than in LDL hypercholesterolemic rabbits. At least three major lipoprotein receptors may be involved in arterial lipid uptake: the LDL receptor related protein, the VLDL receptor and the scavenger receptor(s).28 Both the LDL-receptor-related protein and the VLDL receptor are expressed at the surface of smooth muscle cells in atherosclerotic lesions and may be involved in the direct uptake of ß-VLDL by smooth muscle cells.28–30 In vitro treatment of smooth muscle cells with ß-VLDL induced choles-terol accumulation31 and lysophosphatidylcholine in ß-VLDL induced smooth muscle cell proliferation.32 Oxidized LDL, but not LDL, may also induce smooth muscle cell proliferation.32,33 The uptake of oxidized LDL by smooth muscle cells occurs via scavenger receptors of which expression is regulated by cytokines that are present in atherosclerotic lesions in hypercholesterolemic rabbits.34,35 Thus, the higher rate of progression of atherosclerosis in rabbits with elevated levels of ß-VLDL compared to that in rabbits with elevated levels of LDL may be due to the direct effect of ß-VLDL, independent of oxidation, on smooth muscle cells, whereas LDL can only exert similar effects after oxidation. Those findings may help in understanding the atherogenic role of VLDL in human hyperlipidemia.
Previously, it has been demonstrated that phenotypic modulation and proliferation of smooth muscle cells are key phenomena in human coronary atherosclerosis36,37 and that radical oxidative stress contributes to the progression of cardiovascular disease.38 Recently, we demonstrated oxidized LDL in the coronary artery lesions of ischemic heart disease patients, and we found a strong correlation between plasma levels of oxidized LDL and the extent and the progression of posttransplant coronary artery stenosis in heart transplant patients (submitted for publication). Because of the correlations found between the lesion levels of oxidized ß-VLDL/LDL and the progression of the coronary lesions in the present animal model of coronary atherosclerosis, study of the mechanisms underlying the high sensitivity of coronary arteries to oxidized ß-VLDL/LDL may be possible.
The plasma levels of oxidized apoB-100 containing lipoproteins, ß-VLDL, and LDL, were significantly increased in cholesterol-fed rabbits and in WHHL rabbits and correlated with the total plasma levels of ß-VLDL/LDL. Those findings are in agreement with the previous finding that hypercholesterolemia in rabbits was associated with an increased susceptibility of ß-VLDL and LDL to oxidative modification.39 The correlation between the plasma levels of ß-VLDL/LDL and the lesion amounts of oxidized ß-VLDL/LDL may indicate that either the oxidized ß-VLDL/LDL in the lesions are directly derived from the plasma oxidized ß-VLDL/LDL that infiltrate in the vessel wall or that both the lesion amounts and the plasma levels of oxidized ß-VLDL/LDL reflect similar oxidative stresses both in the vessel wall and in the plasma of hypercholesterolemic rabbits.
In conclusion, the present study in hypercholesterolemic rabbits, both cholesterol-fed NZW and WHHL rabbits, suggests that oxidation of VLDL and LDL that infiltrated in the arterial wall is associated with the progression of coronary atherosclerotic lesions. Although the present data do not allow the conclusion that oxidized VLDL and LDL play a causal role—because the oxidation of those lipoproteins may be a consequence of a more generalized oxidative abnormality, the data are in agreement with a model in which oxidative stress contributes to the progression of cardiovascular disease.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by the Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek (Project 3.0103.92) and by the Interuniversitaire Attractiepolen (P4/34). We thank H. Bernar, E. Brouwers, M. Landeloos, and M. Lox for technical assistance.
Received May 26, 1997; accepted August 7, 1997.
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