© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Relationship Between Lipoprotein- and Oxidation-Related Variables and Atheroma Lipid Composition in Subjects Undergoing Coronary Artery Bypass Graft Surgery
From the Foundation for Blood Research, Scarborough (W.Y.C., S.E.P., L.M.N.); the Maine Medical Center, Portland (M.W.R., C.A.R., E.R., L.M.K.); and The Jackson Laboratory, Bar Harbor (P.M.N.), Me.
Correspondence to Wendy Y. Craig, PhD, Foundation for Blood Research, PO Box 190, Scarborough, ME 04070-0190. E-mail wcraig{at}fbr.org
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Abstract—The relationship between atheroma lipid composition and serum lipoprotein and oxidation measurements has not been fully explored. To address this question, we studied serum, plasma, and aortic wall specimens from 66 subjects undergoing coronary artery bypass graft surgery. The lipid composition of aortic specimens was characterized in terms of cholesterol ester and cholesterol crystal plus phospholipid by using hot-stage polarizing light microscopy; tissue oxidation status was assessed by measuring conjugated dienes. Serum lipoprotein–related measurements included total cholesterol, triglyceride, low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol, apolipoproteins B and AI, and lipoprotein(a). Oxidation status was assessed by measuring LDL mobility, thiobarbituric acid–reactive substances, LDL conjugated dienes, and IgG and IgM autoantibodies against oxidized LDL. Fasting blood glucose was also determined. Lesion cholesterol crystal plus phospholipid content was associated inversely with serum HDL cholesterol levels (r=-0.279, P=0.029) and positively with fasting blood glucose (r=0.359, P=0.016), LDL mobility (0.276, P<0.05), and IgM autoantibodies against oxidized LDL (r=0.272, P=0.037). There was also a significant relationship between the level of aortic tissue conjugated dienes and plasma LDL mobility (r=0.332, P=0.007). In multivariate analysis, IgM autoantibodies against oxidized LDL, fasting blood glucose, and LDL mobility, in descending order of significance, together accounted for 35% of the variability in aortic lesion cholesterol crystal plus phospholipid content. These data support direct and independent roles for oxidation and hyperglycemia in the pathophysiology of atherosclerosis.
Key Words: atherosclerosis • oxidation • lipoproteins • autoantibodies
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Introduction |
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The development of atherosclerosis is a complex, chronic process, which appears to begin as an inflammatory response to vessel wall injury. Atherosclerotic lesions demonstrate characteristic proliferation of smooth muscle cells, an elaboration of extracellular matrix, and the presence of lipid-laden, macrophage-derived, foam cells. In advanced lesions, the core becomes necrotic and lipid is released into the intima, where cholesterol crystals may form.1 2
While it is now well established that certain lipoproteins, most notably LDL, are associated with an increased risk for atherosclerosis, it has also become apparent that LDL modification is necessary before its unregulated uptake by macrophages, and hence foam cell formation, can occur.3 Lipoprotein oxidation is a physiologically relevant form of lipoprotein modification. In vitro studies have demonstrated that oxidized LDL can cause cholesteryl ester accumulation in macrophages4 ; this uptake may occur via the scavenger receptor or by receptors specific for oxidized LDL.5 Oxidized LDL is also a chemoattractant for monocytes and is cytotoxic to endothelial cells, both potentially atherogenic functions (reviewed in Reference 33 ).
The existence of oxidized LDL in vivo is supported by its
detection in atherosclerotic plaque both by immunohistochemical
techniques6 and by direct measurement.7
Furthermore, serum autoantibodies against oxidized LDL (
-oxLDL) have
been detected in humans6 and in animal
models.8 The fact that serum -oxLDL levels are not
correlated with levels of serum lipoprotein– or oxidation-related
analytes suggests that their relevant antigen is not in the plasma
compartment.9 Rather, it appears likely that LDL becomes
oxidized in the subendothelial space, where it has the
opportunity for a prolonged residence time, together with exposure to
the oxygen radicals produced by activated
macrophages.
Data from clinical studies also support the oxidation theory of
atherosclerosis. Stringer et al10 reported
that serum lipid peroxide levels were higher in subjects with
coronary artery disease compared with controls. Similar
findings have been reported for -oxLDL in some,11 12 13 14 15 16
but not all,17 18 19 studies. Recent reports from the
Physicians' Health Study20 21 and the CHAOS (Cambridge
Heart Antioxidant Study) trial22 indicate that a high
dietary intake of vitamin E is associated with decreased risk for
future coronary artery disease. Because vitamin E decreases the
susceptibility of LDL to oxidation,23 these data are also
consistent with an in vivo role for oxidation in the
development of atherosclerosis.
From a clinical standpoint, assessing the degree of lipoprotein
oxidation in an individual might yield important information relevant
to atherosclerosis. Before this approach can be used,
however, several difficulties must be resolved. For example, it has not
yet been established which of the many potential estimates of oxidation
status are the most relevant clinically. Because the analytes of
importance are most likely to be in the vessel wall rather than in the
circulation, it has been suggested that -oxLDL could act as a
surrogate measure of vessel wall oxidation status.24
Direct data to support this approach are lacking. Furthermore, data
relating potential measurements of oxidation status to the clinical and
physiological degrees of
atherosclerosis are scarce.
In the present study, we have begun to address these issues among patients presenting for coronary artery bypass graft (CABG) surgery. Atherosclerotic lesion lipid composition was examined in tissue specimens from the aorta obtained at the time of CABG surgery; fasting plasma and serum specimens for the assay of lipoprotein- and oxidation-related analytes were obtained before surgery. Thus, we were able to examine the in vivo relationship between atheroma lipid composition and levels of circulating analytes related to lipoprotein metabolism and oxidation.
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Methods |
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Subjects
The study group included 66 patients (53 males, 13 females) presenting for elective CABG surgery (
2 vessels). The study was
approved by the Institutional Review Boards at the Foundation for Blood
Research and the Maine Medical Center and was conducted in accordance
with institutional guidelines. Patients were recruited and informed
consent was obtained at the time of the preoperative outpatient visit.
Current vitamin E supplementation, hormone replacement therapy,
antilipemic therapy, or a history of myocardial infarction, angioplasty
or other surgical procedures within the previous 3 months were criteria
for exclusion from the study.
Specimen Handling
Fasting blood samples, obtained on the morning of surgery, were
centrifuged immediately, and serum and EDTA-plasma specimens
were kept at 4°C. When necessary, samples were transported, on ice,
between institutions by courier. As described in more detail below,
assays were performed using either fresh samples or sample aliquots
stored at -70°C. Preservatives (final concentrations: 100 µg/mL
gentamicin, 50 µg/mL chloramphenicol, 1 mmol/L PMSF, 2.7
mmol/L EDTA, 2 mmol/L benzamidine, and 0.01% aprotinin; all from
Sigma Chemical Co) and 1 µmol/L PPACK (Chemica Alta Ltd) were
added to plasma samples before storage at -70°C.
Ascending aorta tissue specimens were obtained during the CABG
procedure. Tissue specimens 
4 mm in diameter were removed from
different locations of the anterior aorta to allow for the insertion of
saphenous vein grafts. The samples were immediately immersed in
ice-cold saline containing 0.1% EDTA and were transferred to the
laboratory for processing. The aortic specimens were frozen rapidly at
-70°C in hexane according to the method of Waugh and
Small25 and kept at -70°C until sectioning. All
specimens were coded, so analyses were blinded as to patient
identity and clinical status.
Histological Studies
Frozen blocks of aorta were mounted on chucks in the cryostat
chamber (Tissue-Tek II, Miles Laboratories Inc) at -25°C with
Cryomatrix (Shandon Southern Instruments, Inc). Sections were cut at
-25°C to a thickness of 5 µm and picked up onto
subbed26 microscope slides for physical and
histological studies. Slides used for the
histological classification of lesion
severity27 were stained with hematoxylin and eosin
(Rapid-Chrome staining kit, Shandon Southern Instruments).
Determination of Tissue Specimen Lipid
Composition
Aortic tissue specimen lipid composition was analyzed
using a combination of hot-stage polarizing light microscopy and
digital image analysis.25 28 Sections to be
examined for lipid content by hot-stage polarizing light microscopy
were unstained and mounted with a drop of glycerol under a No. 1 glass
coverslip (Fisher Scientific). Microscopy was performed on an Olympus
BH-2 microscope fitted with a polarizer, analyzer, and heating
stage control (Rocky Mountain Microscope Corp). Photomicrographs were
taken with a Nikon F3 camera system (Nikon, Inc) with 35-mm film (Kodak
Ektachrome ASA 400).
In brief, unstained aortic sections for physical microscopy were placed on the microscope stage with a heating stage control and viewed under crossed polars. Samples were heated at the rate of 1°C to 2°C/min to 60°C, the temperature above which all cholesteryl esters (CEs) become isotropic.28 29 30 The remaining birefringence represented cholesterol crystal (Cx) plus phospholipid (PL).31 Samples were then cooled on dry ice to 10°C to observe all lesion lipids in crystalline or liquid-crystalline phases. Two sets of photomicrographs were taken at 10°C and 60°C. In the first set, camera exposure time was varied to obtain optimal detail contrast on transparency film; this set was used for visual inspection only. In the second set, the camera exposure time was constant for both temperature settings; this set was used for digital image analysis.
Image processing was performed with custom software written in
PVwave Advantage 5.0 from Visual Numerics Inc (Houston, Tex), a
fourth-generation language for data visualization and processing, run
on a UNIX RISC workstation (DECstation 5000/200, ULTRIX V4.3).
Irregular region-of-interest (ROI) areas were selected by the user via
a custom-designed graphical-user-interface. First, the contours of the
lesion area were outlined by an experienced operator using a computer
mouse. A standard binarization technique was then used, based on visual
inspection of the image, to delineate the areas of lipid birefringence
within the selected ROI. Given a digital image
f(x,y), where x
and y are the spatial coordinates of the image and
0
f(x,y)255 is a
luminance value, a threshold value T was selected such that
0T255. An image pixel was classified as background if
f(x,y)T or as
lipid if
f(x,y)>T. The
total lesion area was obtained by enumeration of all pixels in the ROI,
and the area of lipid birefringence was obtained by enumeration of all
pixels in the ROI with
f(x,y)>T. Area
was calculated from pixel number by scaling with
2, the image plane dimensions of an individual
pixel (in micrometers). The area of lipid birefringence was
expressed as a percentage of total lesion area.
The amount of lipid birefringence quantitated at 10°C represented the lesion total lipids (CE+Cx+PL), whereas that observed at 60°C represented Cx+PL. The birefringence difference between 10°C and 60°C represented CE. All data were expressed as percent of lesion area; data for a given patient represented the average of results from 1 to 4 tissue specimens. The CV for the image processing procedure was 2.6%.
Lipoprotein-Related Assays
Serum cholesterol, triglyceride, and HDL
cholesterol testing (CV<4.4%) was carried out on fresh
samples on a Johnson and Johnson Vitros 700XR analyser with reagents
supplied by the manufacturer. For HDL measurement, dextran
sulfate/MgCl2–selective lipoprotein
precipitation was performed using reagents from Johnson and Johnson.
When serum triglyceride levels were >300 mg/dL, LDL
cholesterol was determined by beta
quantification.32
Apolipoprotein AI and B levels were determined in fresh sera by immunoturbidimetry on a Roche Cobas FARA with antisera and a protocol from INCSTAR and apolipoprotein standard sera from Behring Diagnostics.33 The apolipoprotein assays were calibrated to the International Federation of Clinical Chemistry reference material.34 Within-run and between-run CVs for the apolipoprotein assays were <4%. Lipoprotein(a) [Lp(a)] was assayed in fresh sera by ELISA with kits from Strategic Diagnostics; within-run and between-run CVs were <7.5%.
Lipoprotein Isolation
LDL for oxidation studies (LDL conjugated dienes and LDL
mobility) was isolated from plasma by sequential floatation
ultracentrifugation between densities 1.019 and 1.063
g/mL using the method of Havel et al.35 To minimize
oxidative modification of the sample, all isolation steps were
performed in the presence of 0.01% EDTA and 0.02% BHT.
Oxidation-Related Studies
Several oxidation-related analytes were assayed to encompass a
range of oxidative changes potentially associated with
atherosclerosis. Conjugated dienes were assayed as a
measure of early lipid oxidation and thiobarbituric acid–reactive
substances (TBARS) as an indicator of later aldehyde formation. LDL
mobility was measured because, unlike conjugated dienes and TBARS, the
changes are not reversible and persist through late
oxidation.24 Last, autoantibodies against oxidized LDL
were determined, because these measurements are not confounded by
sample autooxidation and may evaluate the impact of lipoprotein
oxidation.24
To measure LDL mobility, electrophoretic separation of lipoproteins was performed in 1% agarose gels (Chiron) and detected with fat red 7B and Chiron Multitrac lipoprotein kit No. 470695. Duplicate control samples were run in the first and central lanes of each gel. The control material comprised LDL pooled from 3 normolipidemic volunteers, contained 100 µmol/L EDTA and 5 µmol/L BHT to prevent autooxidation, and was stored in aliquots at -90°C. A storage stability study found that control LDL mobility relative to albumin did not differ between fresh samples and those stored for 1, 3, 6, and 9 months at -90°C (data not shown). The mobility of LDL isolated from study subjects was expressed as the relative mobility of the patient sample compared with that of the control, where control migration=1 (assay CV <5%).
For the determination of conjugated dienes, lipids were extracted both from LDL and from the aortic tissue specimens according to the method of Folch et al.36 For tissue analysis, separate samples were taken from the aortic specimens used for histological/lipid composition studies (see above). The level of conjugated dienes was determined by spectroscopic absorbance at 234 nm; data were interpolated from a standard curve, using cholesta-3,5-diene in hexanes (both from Sigma) as the standard. The results were expressed as µmol conjugated dienes/mg cholesterol, with the assumption that cholesterol content reflects the total lipid content of the sample. Plasma cholesterol testing for the lipid oxidation studies was performed using reagents and standards from Sigma. TBARS were assayed, as described previously9 (assay CV=9%), in plasma stored for 2 to 12 months at -70°C in the presence of preservatives (listed above).
IgG and IgM autoantibodies against malondialdehyde-modified LDL
(-oxLDL) were assayed by ELISA in sera stored at -70°C for 2 to
12 months, as described previously,37 with the
modification that NaN3 was omitted from the wash
buffer. In both assays, the antigen (native or malondialdehyde-modified
LDL) was coated at 25 µg/mL and the serum dilution was 1:250. The
dilutions of horseradish peroxidase–conjugated rabbit anti-human IgG
and horseradish peroxidase–conjugated rabbit anti-human IgM (Jackson
ImmunoResearch, Westgrove, Pa) were 1:300 and 1:2000 in the IgG and IgM
assays, respectively. To minimize between-run variability, a serum pool
(n=3 sera) was assayed on each plate. Data were calculated as the
difference in antibody binding between native and
malondialdehyde-modified LDL and then expressed as a percentage of the
value for the serum pool, as described previously.37
Within-run and between-run CVs for the IgG and IgM -oxLDL assays
were 10.5% and 5.6% and for the IgM -oxLDL assay, 5.2% and
10.0%, respectively.
Statistical Methods
Before analysis, analyte data were transformed
logarithmically as necessary, to fit a gaussian distribution. Linear
regression was used to adjust for age and sex. Bivariate relations
between variables were examined by linear regression and
multivariate relations by stepwise linear regression.
Significance was accepted at P<0.05. All analyses
were performed using a statistical package from BMDP Statistical
Software, Inc.
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Results |
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Descriptive Data
Demographic, lipoprotein, and fasting blood sugar data are presented for all subjects, according to sex, in Table 1
. The study group included 64 subjects
for whom vessel wall data were available. There were no subjects with
insulin-dependent diabetes mellitus in the study group and 14 subjects
(6 females, 8 males) with non–insulin-dependent diabetes mellitus. The
frequency of self-reported smoking was similar in men (22.6%, 12/53)
and women (23.0%, 3/13).
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Lesion lipid data for a given subject were available from 1 (n=15
subjects), 2 (n=31 subjects), 3 (n=17 subjects), or 4 (n=1 subject)
tissue specimens. Atheromatous lesion was observed in
97% of the 132 histological tissue specimens examined;
among lesions, 1.6% were Stary classification type I, 9.6% were type
II, 40% were type III, 47.2% were type IV, and 1.6% were type
V.17 Variations in lipid lesion composition between aortic
samples within an individual, calculated as the within-person CV, were
38% for Cx+PL, 47% for CE, and 27% for total lipid. Lesion lipid
data are shown in Table 2. Within
lesions, there was no relationship between the content of CE and Cx+PL
(n=63, r=0.105, P=0.414). Table 3 shows oxidation-related data for plasma
or serum and for aortic tissue; of the measured variables.
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Relationships of Serum Parameters With
Atheroma Lipid Composition and Oxidation Status
Serum fasting blood glucose levels were positively associated with
aortic lesion levels of both Cx+PL (r=0.359,
P=0.016, n=45) and total lipid (r=0.319,
P=0.033, n=45). Cx+PL also demonstrated a significant
inverse relationship with serum HDL cholesterol level
(r=-0.279, P=0.029, n=61) and a similar, but not
significant, inverse relationship with apo AI, the major protein moiety
of HDL (r=-0.223, P=0.077, n=64). There were no
relationships between other serum lipid and lipoprotein levels [total
cholesterol, triglycerides, Lp(a), LDL
cholesterol, and apo B] and lesion total lipid, CE, or
CE+PL. Furthermore, serum lipid and lipoprotein levels were not
associated with aortic tissue oxidation status, as estimated by the
measurement of conjugated dienes.
The relationships between atheroma lipid composition
and indicators of plasma oxidation status are shown in Table 4. There were no associations of the
measured plasma oxidation variables with lesion CE; however, levels
of LDL conjugated dienes, an "early" oxidation product, were
significantly associated with lesion total lipid, whereas LDL mobility,
a marker for more advanced modification, was associated with lesion
Cx+PL content. Autoantibodies against oxidized LDL demonstrated
associations with both lesion Cx+PL and total lipid; the associations
were strongest for IgM -oxLDL. In contrast, plasma levels of TBARS,
a relatively nonspecific measure of lipid oxidation, had no association
with lesion lipid composition. The same results were obtained when
these bivariate analyses were performed in nondiabetic subjects
only (data not shown). LDL mobility, a measure of plasma oxidation
status, was associated not only with atheroma lipid
composition but also with aortic tissue conjugated dienes, a measure of
tissue oxidation status (n=64, r=0.332,
P=0.007).
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Independent Predictors of Variability in Atheroma
Lipid Composition
Lesion Cx+PL content was used as a surrogate measure of lesion
severity.17 All variables that predicted at least 4%
of variability in Cx+PL levels in bivariate analysis
(r0.200) were included in a multivariate
model to identify independent predictors of variability in Cx+PL; these
included HDL cholesterol, apo AI, fasting blood glucose,
LDL mobility, IgM -oxLDL, and IgG -oxLDL. As shown in Table 5, IgM -oxLDL, fasting blood glucose,
and LDL mobility, in descending order of significance, were independent
predictors of Cx+PL levels, together accounting for 35% of the
variability in lesion Cx+PL.
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Discussion |
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Considerable evidence to support a role for oxidative processes in the development of atherosclerosis exists, although much remains to be learned about the specific mechanisms involved.38 Furthermore, the clinical utility of measuring oxidation status has yet to be fully explored, both in terms of potential analytes and their relevance to clinical populations. The present study addresses these questions by examining the associations between measurable serum parameters of oxidation status and the lipid composition and oxidation status of atherosclerotic plaque. The fact that multiple comparisons can give rise to statistical significance simply due to chance should be kept in mind when interpreting the results of this study.
The use of an in vivo human model presents certain experimental difficulties. Lesion data were obtained using tissue specimens obtained during CABG surgery, rather than the complete arterial specimens available in animal studies. Our approach assumes that the tissue samples obtained were representative of aortic atherosclerosis status in a given patient. Because multiple samples from the aortic arch were available for most subjects, it was possible to assess the extent of within-subject heterogeneity. Given that within-subject variation in lesion lipid composition was evident, our data most likely underestimate the association of these parameters with other measured variables.
The uptake of CE into the lysosomes of macrophages, followed by its conversion to free cholesterol by acid cholesteryl ester hydrolase, leads to the accumulation of free cholesterol in the lysosome and crystallization of cholesterol monohydrate.39 Interactions between foam cell intracellular Cx and CE pools are not, however, well understood. We found no association between the respective levels of Cx and CE in the aortic lesions. This may indicate either that these 2 pools are regulated separately or that interactions between the 2 pools are not in equilibrium.
Lipid composition contributes to the mechanical properties of plaque40 and thus may influence plaque stability; plaque rupture and consequent thrombosis is an important determinant of the acute occlusive event in cardiovascular disease.1 2 Soft plaque with a high CE content is believed to be less stable than a mechanically stiffer plaque characterized by the presence of Cx.41 In the present study, the associations between serum or plasma markers and lesion lipid composition were all with lesion Cx content rather than with CE. Thus, the analytes measured may be more important as markers for plaque development than for plaque instability.
The finding of inverse relationships between HDL
cholesterol or apo AI levels and lesion Cx content
(significant for HDL cholesterol) is consistent
with the results of epidemiologic studies that have demonstrated a
protective role for the HDL particle in
atherosclerosis.42 The relationships were
not significant in multivariate analysis,
suggesting an interaction between HDL and other variable(s) in the
model. With the exception of HDL cholesterol, other
traditional lipoprotein measurements demonstrated no significant
associations with lesion lipid composition. In contrast, 2
oxidation-related variables, LDL mobility and IgM -oxLDL, were
significantly and independently associated with lesion Cx content,
providing direct support for the hypothesis that the LDL particle only
becomes atherogenic after chemical modification. Furthermore, the
association between LDL mobility and aortic tissue oxidation status, as
estimated by conjugated dienes, suggests a direct relationship between
oxidation processes in these 2 compartments. This relationship may be
explained by the uptake of circulating modified lipoprotein by the
arterial wall, though other possibilities, such as a common
arterial oxidative process followed by migration of
modified LDL from the subendothelial space back into
the circulation, cannot be ruled out. Further work is needed to define
this relationship. For example, both the composition of oxidized lipids
and their extent of oxidation are likely to be
heterogeneous in plasma and tissue, and additional
oxidation assays may be needed for the optimal estimation of oxidation
status.
There are at present inconsistencies in the literature
regarding the relationship between -oxLDL and
atherosclerosis.11 12 13 14 15 16 17 18 19 Most previous
studies have focused on measurements of IgG -oxLDL rather than IgM
-oxLDL. As demonstrated here, there is a relationship between IgG
-oxLDL and lesion lipid content, but it is weaker than the
relationship observed for IgM -oxLDL and could be easily confounded
by the presence of subclinical atherosclerosis in
control groups. There are also conflicting data, however, when
atherosclerosis is ascertained by imaging techniques.
For example, using angiography, Van der Vijver et al43
found no significant relationship between -oxLDL and the degree of
stenosis, whereas Salonen et al44 demonstrated a
significant association when atherosclerosis was
quantified as intima-media thickness of the carotid artery. The type of
-oxLDL measured may explain this discrepancy; however, -oxLDL may
also have different relationships with the physical characteristics of
atherosclerosis as detected by imaging techniques,
compared with the compositional characteristics as presented
here.
Diabetes is an independent risk factor for atherosclerosis and is associated with a 2- to 4-fold increased risk for this disease.45 46 Although an adverse lipoprotein profile may account for some of this excess risk, particularly in non–insulin-dependent diabetes mellitus, there has been recent interest in the role of hyperglycemia in the pathophysiology of atherosclerosis.47 Consistent with a direct effect, we found a significant relationship between fasting blood glucose and lesion Cx content in multivariate analysis, indicating that blood glucose level contributes independently to the development of atherosclerosis. These findings are consistent with the data of Litvak et al,48 who reported that chronic hyperglycemia was associated with increased atherosclerosis in monkeys, in the absence of hyperlipidemia. Also, McGill et al49 found in autopsy studies of young males that subjects with glycohemoglobin levels >8% had more extensive coronary and aortic atherosclerosis than did those with lower levels.
In summary, we have examined the in vivo relationships between
atherosclerotic lesion lipid composition and serum or plasma
measurements of lipoprotein- and oxidation-related variables. LDL
mobility, IgM -oxLDL, and fasting blood glucose were significant
independent predictors of lesion Cx+PL content, accounting for a large
portion (35%) of the variability in this analyte. These data support
the existence of direct roles for oxidation and hyperglycemia in the
pathophysiology of atherosclerosis.
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Acknowledgments |
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This work was supported by National Institutes of Health grant 5 P20 RR10203-02 (to P.M.N.) and Department of Energy grant DE-FGO293ER75945 (to W.Y.C.). The authors would like to thank Mariusz Jankowski, PhD, for assistance with the image processing studies and Agatha Bellevue, RN, for assistance with patient recruitment.
Received May 28, 1998; accepted November 25, 1998.
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