Increased Apolipoprotein Deposits in Early Atherosclerotic
Lesions Distinguish Symptomatic From
Asymptomatic Patients
Moritz Wyler von Ballmoos, Denise Dubler, Martina Mirlacher, Gieri Cathomas,
Jürgen Muser, Barbara C. Biedermann
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Objective—Apolipoprotein E (apoE) and apolipoprotein B100 (apoB) are both involved in receptor-mediated uptake of
atherogenic lipoproteins by the liver. Inefficient hepatic clearance of these lipoproteins leads to symptomatic
atherosclerosis. Using arterial tissue microarrays, we tested the hypothesis that apoE and apoB accumulation in the
arterial wall discriminates between patients with symptomatic atherosclerosis and patients who never experienced
cardiovascular events.
Methods and Results—In a postmortem study involving 49 patients (22 patients with symptomatic atherosclerosis), we
quantified apolipoprotein deposits in arterial rings obtained from the left main coronary, the common carotid, the
common iliac, and the renal artery applying tissue microarray technology and semiquantitative immunohistochemistry.
In early atherosclerotic lesions, even before atheroma appeared, symptomatic patients had significantly more arterial
apoE and apoB deposits than patients without cardiovascular events (P⬍0.001). Among the symptomatic patients, those
without diabetes had more intense apolipoprotein deposits than diabetics. Large amounts of apoE and apoB were found
in advanced atherosclerotic lesions, regardless of the activity of the disease.
Conclusions—Increased apolipoprotein deposits are an early sign of symptomatic atherosclerosis. They may reflect either
enhanced retention of atherogenic lipoproteins or impaired local apolipoprotein degradation. The arterial lipoprotein
turnover may be different in diabetic patients. (Arterioscler Thromb Vasc Biol. 2006;26:359-364.)
Key Words: arteriosclerosis 䡲 lipoproteins 䡲 apolipoproteins 䡲 diabetes mellitus 䡲 tissue microarray
A
therosclerosis is an inflammatory lipid storage disease
affecting large and medium-sized arteries.1–3 Abnormal
lipoprotein retention in the arterial wall has been described as
one of the earliest steps in the development of atherosclerotic
lesions.4 – 6 The apolipoproteins are the protein part of the
lipoprotein particles and play an important role in the interaction of lipoproteins with cells and the extracellular matrix.
Apolipoprotein E (apoE) and apolipoprotein B100 (apoB) are
synthesized in the liver and other organs. Both apoE and
apoB mediate low-density lipoprotein (LDL) receptor–mediated uptake of lipoproteins by the liver, the prerequisite of
cholesterol elimination from the body.7 Mutations of the
apoB and apoE gene, which affect LDL receptor binding,
lead to familial hypercholesterolemia8 or dysbetalipoproteinemia.9 Both conditions are associated with premature
atherosclerosis because of an impaired hepatic clearance of
atherogenic lipoproteins. Intimal lipoprotein retention has
been shown to accelerate atherosclerosis in an animal model
of the disease,5 and extensive intimal lipid accumulation is a
structural hallmark of the advanced and vulnerable atherosclerotic lesion.10,11 However, although intimal lipoprotein
accumulation is known to precede plaque formation,12 little is
known about the significance of early lipoprotein retention
for the development of symptomatic atherosclerosis in humans. We have developed the human arterial tissue microarray technique to identify morphological signs of common
pathways, which lead to unstable or vulnerable plaques.13
Forty percent to 50% of patients who are treated in our
hospital have experienced cardiovascular events during their
lifetime and fulfill the definition of symptomatic atherosclerosis. Therefore, the prospective inclusion of hospitalized
patients in a nonselective manner assembles a cohort of
patients that is highly representative for the population that
this hospital is serving. We have successfully used the tissue
microarray technique to systematically investigate the arterial
wall at different lesional stages in patients with symptomatic
atherosclerosis.13 We described previously the typical pattern
of neovascularization and inflammation in vulnerable patients
Original received August 16, 2005; final version accepted November 10, 2005.
From the Department of Medicine (M.W.v.B., D.D., J.M., B.C.B.), University Hospital Bruderholz, Bruderholz, Switzerland; Department of Research
(M.W.v.B., D.D., B.C.B.), University Hospital Basel, Basel, Switzerland; Institute of Pathology (M.M.), University of Hamburg-Eppendorf, Hamburg,
Germany; and Cantonal Institute of Pathology (G.C.), Liestal, Switzerland.
Correspondence to Barbara C. Biedermann, Department of Medicine, University Hospital Bruderholz, Batteriestrasse 1, CH-4101 Bruderholz,
Switzerland. E-mail [email protected]
© 2006 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
359
DOI: 10.1161/01.ATV.0000198250.91406.6d
360
Arterioscler Thromb Vasc Biol.
February 2006
Patient Characteristics, Tissue Selection, and Plaque Burden
No Cardiovascular Events
(n⫽27 patients)
Cardiovascular Events
(n⫽22 patients)
P Value
Female sex, n (%)
14 (52)
11 (50)
0.90
Age, y
74⫾14
79⫾9
0.09
Arterial hypertension
6 (22)
11 (50)
0.04
⬍0.001
Characteristic
Patients with cardiovascular risk factors, n (%)
Diabetes mellitus
1 (4)
10 (45)
Hypercholesterolemia
2 (7)
7 (32)
0.03
Smoking
5 (18)
7 (32)
0.35
Arterial sectors analyzed by tissue microarrays, n
Total, all sites
202
148
Common carotid artery
54
44
Left coronary artery
40
16
Renal artery
54
44
Common iliac artery
54
44
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Frequency distribution of atherosclerotic plaques, n (%)
AHA plaque type 0
21 (10)
10 (7)
AHA plaque type 1
61 (30)
33 (21)
AHA plaque type 2
58 (29)
41 (28)
AHA plaque type 3
34 (18)
20 (14)
AHA plaque type 4
14 (7)
15 (10)
AHA plaque type 5
7 (3)
17 (11)
AHA plaque type 6
7 (3)
12 (8)
with symptomatic atherosclerosis, that is, in patients who
experienced cardiovascular disease during their lifetime.13 In
the present analysis, we used the tissue microarrays to
investigate the pattern and extent of apolipoprotein deposits
in the arterial wall of vulnerable patients and compared these
findings with asymptomatic patients who did not develop
cardiovascular events during their lifetime.
Methods
Arterial Tissue Microarrays
All of the investigations were approved by the institutional ethical
review board. Between December 2002 and April 2003, 49 patients
who died at our hospital (Department of Medicine, University
Hospital Bruderholz, Bruderholz, autopsy rate ⬎75%) were consecutively included in this study. Twenty two of the 49 patients
experienced cardiovascular events (eg, myocardial infarction, stroke,
or peripheral arterial occlusive disease) during their lifetime, that is,
they had symptomatic atherosclerosis. The other 27 patients had no
cardiovascular events, that is, they were asymptomatic with respect
to atherosclerosis. The clinical characteristics of these patients are
summarized in the Table.13 At autopsy, a 0.5-cm–long ring segment
of the left common carotid, the left renal, and the left common iliac
artery was removed, fixed immediately in 4% phosphate-buffered
formalin (pH 7.4), and embedded in paraffin. When acute coronary
heart disease was suspected, a postmortem coronary angiography
was routinely performed. Therefore, the left main coronary artery
segment, which was not treated equally for all patients, was obtained
only from the 28 patients who did not undergo postmortem angiography. The arterial tissue microarrays from the iliac, the renal, and
the carotid arteries have been described previously, and the microarrays from the coronary arteries were constructed in a similar way.13
In brief, the complete arterial ring sections were stained with
Elastica-van Gieson and examined histologically for the presence of
atherosclerotic lesions. From each arterial ring, the best preserved
and the most affected arterial sectors were typed for the presence of
atherosclerotic plaques according to the American Heart Association
(AHA) consensus report.10,14,15 The typing results were used for the
assessment of plaque burden (Table; Figure 2A). The 2 sectors were
punched out from the donor paraffin block and transferred to a
recipient paraffin block as described.13 Skin, tonsil, liver, and kidney
were generally included in the microarray blocks as control tissues.
Immunohistochemistry
Six-␮m–thick sections of the microarray block were cut and transferred to glass slides. The sections were air dried (60°C, 45 minutes),
paraffin was removed by xylol, and the slides were hydrated.
Endogenous peroxidase activity was blocked by 2% H2O2. For
antigen retrieval, slides were either incubated for 2 minutes in
10 mmol/L citrate buffer in a steam pressor chamber for apoE
staining or treated with proteinase K (S3020, DAKO, Zug, Switzerland) for apoB staining. Slides were rinsed in PBS and incubated
with the first antibody: polyclonal goat anti-human apoB (ab7616,
Abcam, Cambridge, UK) or monoclonal mouse anti-human apoE
(SIG-9740-02, Alexis Biochemicals, Lausen, Switzerland). After 60
minutes of incubation at room temperature, the slides were washed
twice and incubated with a peroxidase-conjugated rabbit anti-goat
antiserum (Universal Quick kit, Novocastra, Newcastle-on-Tyne,
United Kingdom) or goat anti-mouse antiserum (Envision system
HRP mouse, DAKO). After 30 minutes of incubation, slides were
washed twice and incubated with diaminobenzidine as a substrate.
Hemalaun was used for counterstaining before the sections were
dehydrated and embedded (Pertex, Medite, Nunningen,
Switzerland).
Semiquantitative Assessment of Apolipoprotein
Deposits in the Tissue Arrays
ApoE and apoB were detected in the extracellular matrix of the
intima and, to a lesser extent, in the media layer of the arteries. In
most of the microscopically healthy appearing arterial sectors,
apolipoproteins were generally not detectable, and the amount of
Wyler von Ballmoos et al
Apolipoprotein Deposits in Atherosclerosis
361
Coulter Inc) according to the manufacturer’s instructions. The band
intensity of apoB as quantified by Western blotting correlated
linearly with the amount of apoB measured nephelopmetrically
(R⫽0.93; P⬍0.001).
Statistics
The statistical analyses were performed using SPSS 11.0 software
(SPSS Inc). Plaque burden and apolipoprotein staining scores between asymptomatic and vulnerable patients and apoE and apoB
staining scores at the different stages of the disease were compared
by Mann-Whitney U test. The interobserver correlation was analyzed
by the Spearman’s rank test. P values ⬍0.05 indicated significant
differences. Unless otherwise stated, median values and the interquartile range are shown.
Results
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Figure 1. The semiquantitative scoring system used to quantify
apolipoproteins in the arterial intima. (A through D) Typical
examples for the semiquantitative staining score. The red dotted
line marks the border between intima (I) and media (M). Scale
bars represent 100 ␮m.
deposited apolipoproteins gradually increased with increasing severity of atherosclerotic lesions (Figure 1A through 1D). To quantify the
intimal apolipoprotein deposits by immunohistochemistry staining,
the following semiquantitative scoring system was applied: 0 indicates no apolipoprotein deposits; 1, fine, mostly linear deposits in the
intima (Figure 1A); 2, multilamellar deposits in the intima, ⬍50% of
intima area (Figure 1B); 3, confluent deposits covering ⬎50% of the
intima area (Figure 1C); and 4, the intima is nearly completely filled
with apolipoprotein deposits (Figure 1D). The interobserver variability of this scoring system was assessed by the comparison of the
staining scores obtained independently by 2 investigators (M.W.v.B.
and B.C.B.). Because apoE and apoB were deposited similarly in the
arterial wall, the same scoring system was applied for both
apolipoproteins.
Quantification of Human Plasma Apolipoproteins
Human EDTA plasma was obtained from 26 patients (10 without a
history of cardiovascular events and 16 with symptomatic atherosclerosis) after written informed consent was given. The plasma was
diluted 1:10 in PBS and 1:5 in reducing sample buffer. ApoE was
separated on a 10% and apoB on a 6% SDS-PAGE, respectively. The
proteins were transferred in transfer buffer (25 mmol/L Tris base;
Merck), 192 mmol/L glycin (Fluka), and 20% pure methanol to
cellulose nitrate (Protran BA83, Schleicher&Schüll). After washing
the membrane in Tris-buffered saline, nonspecific protein binding
sites were blocked for 60 minutes in 5% fat-free milk. The membrane was then incubated with the first antibody for 60 minutes at
room temperature. After extensive washing in Tris-buffered saline,
the membrane was transferred either to the peroxidase-conjugated
secondary rabbit anti-goat antibody (P0449, DAKO) or to the
peroxidase-conjugated goat anti-mouse antibody (115-035-071,
Jackson Laboratories, Bar Harbor, Maine) and incubated for 60
minutes at room temperature. The membrane was washed again and
incubated with chemiluminescence substrate (Supersignal West
Pico, Pierce, Perbio Science). A photographic film (Biomax, Eastman Kodak) was exposed to the membrane and developed using an
automatic processor (Crurix 60, Agfa). A molecular weight standard
[Color Marker (wide range), ␴-Fluka] was used to calibrate the
electrophoretic separation of plasma proteins. Band intensity was
measured with an image analysis software (ImageJ, please see online
at http://rsb.info.nih.gov/ij/, National Institutes of Health) and expressed in relative units. ApoE and apoB concentrations were
normalized to normal pool plasma (set to be 100%). ApoB concentrations in the normal pool plasma were quantified by immunonephelometry on the Beckman Nephelomatic Image 800 (Beckman-
Histological Analysis of Apolipoprotein
Accumulation in the Arterial Wall
Fully developed and advanced atherosclerotic lesions (AHA
type 4 to 6) showed extensive deposits of apoE and apoB
covering most of the intima area (Figure 1D). The distribution
of apoE and apoB in the atherosclerotic plaques was indistinguishable and, therefore, the same staining score was used
to quantify both apolipoproteins. Both apoE and apoB are
synthesized in the liver. The liver tissue, which was included
as a control in each of the tissue microarray blocks, displayed
hepatocellular staining for both proteins (data not shown).
ApoE is also synthesized and secreted by macrophages.16
However, in the tonsillar tissue, which is included as a control
tissue and in the arterial adventitia, which contains macrophages, apoE deposits could not be detected. The specificity
of the antibodies was confirmed by Western blotting of
human plasma to rule out nonspecific cross-reactivity of the
antibodies that were used for immunohistochemistry. The
anti-apoE and the anti-apoB antibody both detected a single
protein band of the expected size (32 kDa and 500 kDa,
respectively).
Interobserver Correlation of the Apolipoprotein
Staining Score
The amount of apolipoproteins in the intima at early stages of
the disease was assessed by a semiquantitative scoring system
(Figure 1A through 1D). The interobserver correlation for this
scoring system was high (y⫽0.96x ⫹0.2, r⫽0.87, P⬍0.001
for apoE and y⫽0.94x ⫹0.3, r⫽0.85, P⬍0.001 for apoB). In
fully developed atherosclerotic lesions, apolipoproteins were
found most abundantly in the center of the plaques. A cellular
association with endothelial, fibromuscular, or inflammatory
cells was not detected.
Arterial Apolipoprotein Deposits Are a Panarterial
Sign of Atherosclerosis
The apolipoprotein deposits were quantified in asymptomatic
patients at all 4 of the arterial sites, that is, the renal, the
common carotid, the common iliac, and the left main coronary artery. The tissue arrays that were used for apolipoprotein staining contained a substantial number (247 of a total of
350 sectors) of arterial sectors with early atherosclerotic
lesions (AHA type 1 to 3) and even 31 sectors without any
pathologic changes of the arterial wall (Figure 2A; Table).
Therefore, the gradual increase of apolipoprotein deposits in
362
Arterioscler Thromb Vasc Biol.
February 2006
Figure 3. Apolipoprotein deposits in the arterial sectors from
patients with (n⫽22) and without (n⫽27) cardiovascular events.
Arterial sectors from symptomatic (148) and arterial sectors from
asymptomatic patients (202) were analyzed. For both patient
groups, apoE (A) and apoB (B) deposits at early (AHA plaque
type 0 and 1), intermediate (AHA plaque type 2 and 3), and
advanced (AHA plaque type 4 to 6) lesions are shown.
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Figure 2. Plaque burden and arterial apolipoprotein deposits in
asymptomatic patients. (A) The number of arterial sectors per
plaque category10 is shown for the 4 blood vessels analyzed.
ApoE (B) and apoB (C) deposits in patients without cardiovascular events, that is, patients with asymptomatic atherosclerosis.
n.a. indicates, in the renal artery of asymptomatic patients,
advanced plaque types were not available for scoring.
the subendothelial area of the intima could be reliably
analyzed (Figure 2B and 2C). At all 4 of the arterial sites, no
or minimal apolipoprotein deposits were detectable in normal
arterial sectors or in sectors with intimal thickening (AHA
type 0 or 1), multilamellar apolipoprotein deposits at sites
with fatty streaks, or preatheroma (AHA type 2 or 3) but
uniformly extensive apolipoprotein deposits covering nearly
100% of the arterial intima in fully developed atheroma and
advanced lesions (AHA type 4 to 6). Therefore, apoE and
apoB retention are panarterial signs of atherosclerosis. The
iliac and the coronary arteries both have a higher plaque
burden than the renal and carotid artery (Figure 2A). However, the amount of apolipoproteins accumulating in these
arteries is determined by the severity of atherosclerotic
lesions at all 4 of the arterial sites (Figure 2B and 2C) and not
by the plaque burden of the examined vascular bed.
scopically healthy appearing arterial wall or in intimal thickening (AHA plaque type 1), symptomatic patients had a
higher apoB and apoE staining score than asymptomatic
patients (staining score 2 [range, 1 to 3] versus 1 [range, 0 to
1], P⬍0.001, for apoB and 1 [range, 1 to 2] versus 0 [range,
0 to 1], P⬍0.001, for apoE, respectively; Figure 3). At
intermediate atherosclerotic lesions such as fatty streaks
(AHA plaque type 2) or preatheroma (AHA plaque type 3),
symptomatic patients had still higher apoB deposits than
asymptomatic patients (staining score 3 [range, 3 to 3] versus
2 [range, 2 to 3], P⬍0.001; Figure 3), but apoE deposits were
not significantly different. Elevated plasma apolipoprotein
levels could explain the increased deposits in vulnerable
patients. However, plasma apoE and apoB levels were not
significantly different between the 2 groups of patients
(Figure 4).
Diabetes mellitus is a known risk factor for atherosclerosis,
and we tested whether, among the symptomatic patients,
patients with diabetes mellitus would have increased apolipoprotein deposits. Unexpectedly, vulnerable patients with
diabetes mellitus had significantly less apoB and apoE
deposited in the arterial wall at early stages of atherosclerosis
compared with patients without diabetes [staining score 1
(range, 0.9 to 2) versus 2.5 (range, 1.75 to 3), P⫽0.02, for
Arterial Apolipoprotein Deposits Are an Early
Sign of Symptomatic Atherosclerosis
We next analyzed whether apolipoprotein accumulation was
different in symptomatic patients who experienced cardiovascular events and asymptomatic patients. Vulnerable patients
had a significantly higher plaque burden [average AHA
plaque type 2.7 (range, 2.1 to 3.3) versus 2.2 (range, 1.9 to
2.6); P⫽0.01] and significantly more apoE and apoB deposits
[staining score 2.6 (range, 2.2 to 3.0) versus 2 (range, 1.25 to
2.1), P⫽0.002 for apoE and 3 (range, 2.7 to 3.3) versus 2
(range, 1.3 to 2.4), P⬍0.001 for apoB] compared with
patients who never experienced cardiovascular events. When
arterial sectors with similar plaque types were compared,
vulnerable patients had significantly more apolipoproteins
deposited at the earliest stages of the disease. In the micro-
Figure 4. Plasma levels of apoE and apoB in patients with
(n⫽16) and without (n⫽10) cardiovascular events.
Wyler von Ballmoos et al
apoB and 1 (range, 0.5 to 1.1) versus 2 (range, 1 to 2.1),
P⫽0.02, for apoE, respectively]. Nonenzymatic glycation
could modify the antigenic structure of apolipoproteins,
thereby reducing the avidity of the antibodies used for
immunohistochemistry. To rule out that antibody binding was
impaired by any posttranslational mechanism precipitated by
diabetes mellitus, we performed Western blot analysis of
plasma apoE and apoB in 3 diabetic patients with elevated
HbA1c (⬎10%) and compared it with nondiabetic control
patients. The staining intensity of the apolipoprotein bands
was not influenced by posttranslational modification, such as
nonenzymatic glycation (data not shown). Therefore, reduced
antibody binding to modified apolipoproteins in diabetic
patients with poor metabolic control cannot explain the
modest staining of apolipoproteins in the arterial intima.
Discussion
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In this tissue microarray analysis of arterial apolipoprotein
deposits, we found that vulnerable patients, that is, patients
who experienced symptomatic atherosclerosis during their
lifetime, have increased apoE and apoB deposits at the
earliest lesional stages, that is, during intimal thickening or
even in microscopically healthy arteries. The histomorphological pattern of apolipoprotein distribution in the arterial
wall was nearly identical for apoE and apoB. As an unexpected finding, patients with diabetes mellitus had minimal
intimal apolipoprotein deposits in early atherosclerosis as
detected by immunohistochemistry.
The net amount of apolipoproteins in the arterial wall is the
sum of delivery or influx, local synthesis or retention, and
degradation or efflux. Our finding that apolipoproteins first
accumulate in the arterial intima, that media deposits are
found only when extensive intimal deposits are present, and
that apolipoproteins are virtually absent from the adventitia
supports the concept of a centrifugal influx of apolipoprotein
from the luminal side of the arterial blood vessel. We have
observed a similar, centrifugal deposition of von Willebrand
factor, another large molecule, in the extracellular matrix of
large arteries (data not shown). ApoE is known to be
synthesized by macrophages, particularly, activated lesional
macrophages in atherosclerosis,16 and is supposed to mediate
reverse cholesterol transport.17,18 However, we had no evidence that apoE would show cell-bound macrophage or foam
cell staining, nor that it would be preferentially deposited
around macrophage-rich areas of the atherosclerotic plaques.
These findings either suggest that apoE, which is synthesized
locally and which serves as an acceptor of cholesterol
secreted by macrophages, is not retained locally but rapidly
removed from the arterial wall in the course of an efficient
reverse-cholesterol transport pathway. As an alternative explanation, the amount of locally produced apoE could be too
low to be detected by immunohistochemistry. Transendothelial influx is not only determined by endothelial dysfunction
but also by lipoprotein particle size.19 Indeed, small LDL
particle size has been associated with symptomatic atherosclerosis.20 In addition to the binding sites for lipoprotein
receptors, apolipoproteins have specific binding sites for
extracellular matrix components, such as proteoglycans. Proteoglycans (eg, versican, biglycan, decorin, perlecan, etc)
Apolipoprotein Deposits in Atherosclerosis
363
contain negatively charged glycosaminoglycan chains, which
preferentially bind to basic amino acids. The proteoglycan
binding site of apoB is located in a cluster of basic amino
acids between residue 3359 and 3369,21 and the active
binding site of apoE is located between amino acids 142 and
147.22 The mutational analysis of the proteoglycan binding
sites of human apoB has demonstrated in several transgenic
mouse lines that reduced matrix retention of LDL in the
arterial wall was associated with diminished formation of
atherosclerosis.5 Similarly, apoE is directly involved in the
retention of lipoproteins to the arterial wall.23 ApoB and apoE
are components of different lipoproteins (chylomicron remnants, LDL, very low– density lipoprotein, intermediatedensity lipoprotein, and high-density lipoprotein), which
exert proatherogenic and antiatherogenic effects. The similar
pattern of apoB and apoE deposits and the close association
with symptomatic atherosclerosis suggest that, in the arterial
wall, both apolipoproteins are tracing the local delivery of
proatherogenic lipoproteins. Finally, reduced cellular uptake
and degradation by resident arterial cells, such as macrophages and smooth muscle cells, could also explain our
observational data. We have shown previously that macrophages are present in early stages of atherosclerotic lesions13
and, therefore, cells would be locally available for the
phagocytosis of lipoprotein particles. The extracellular pattern of apolipoprotein deposits found in the human arterial
wall suggests that, with the progression of lesion formation,
cellular uptake and degradation of apolipoproteins are increasingly ineffective to remove these proteins from the
arterial intima.
The fact that patients with diabetes have less apolipoprotein deposits in the arterial wall is an intriguing finding of this
study. Diabetes mellitus is a known risk factor for symptomatic atherosclerosis. In fact, 10 of 11 diabetic patients in our
cohort are in the group with symptomatic disease.13 Endothelial dysfunction is a hallmark of diabetic macroangiopathy
and microangiopathy. Therefore, diminished transendothelial
influx may not explain the reduced level of apolipoprotein
deposits in these patients. Poor metabolic control and hyperglycemia may facilitate nonenzymatic glycation of extracellular matrix proteins and, therefore, reduce avidity of apolipoproteins for proteoglycans.24 We showed by Western
blotting that immune recognition of apoB and apoE by the
antibodies used in this study is not affected by nonenzymatic
glycation or other posttranslational modifications occurring
in the blood. Diabetic patients have preferentially small LDL
particles,25 and these atherogenic lipoproteins have been
shown to increase binding and uptake by monocyte-derived
macrophages.26 Our data could be explained as follows: in
diabetic patients, scavenger receptor–mediated uptake of
intimal lipoproteins is enhanced, and, therefore, intimal
apolipoprotein deposits are reduced. Because LDL uptake
and foam cell formation27 are c-Jun kinase 2– dependent
processes, this signaling pathway may be activated during the
pathogenesis of diabetic macroangiopathy. Selective uptake
of cholesterol esters leaving back apolipoproteins extracellularly28 could be the favorite pathway of lipoprotein uptake in
nondiabetic patients. Because we cannot definitely exclude
that modifications of apolipoproteins in situ, by oxidation, or
364
Arterioscler Thromb Vasc Biol.
February 2006
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by other mechanisms that are exclusively active in the arterial
wall could explain the low immunohistochemical staining
observed in diabetic patients, our findings need to be interpreted with caution.
The experimental approach described here is limited by the
fact that, in this postmortem study, early lesional stages from
vulnerable patients were analyzed after cardiovascular events
occurred. Prospective data that would support the concept
that incipient apolipoprotein deposits in the arterial wall
identify presymptomatic patients who will develop symptomatic atherosclerosis later in their life are difficult to obtain.
Despite this limitation, our findings encourage the use of
lipoproteins containing apoE or apoB as targeting devices to
deposit radionuclids or drugs to the diseased arterial intima,
precisely to the site where atherosclerosis starts. In the
nondiabetic subset of patients, this may offer a promising
diagnostic or therapeutic approach to reach not only advanced
lesions but also healthy appearing arteries or, in the context of
secondary prevention, less affected vascular beds.
12.
13.
14.
15.
16.
Acknowledgments
This work was supported by a grant from the Swiss National Science
Foundation (3200-664121) and by a Specific Targeted Research or
Innovation Project (MOLSTROKE) of the 6th Framework Programme of the European Union.
17.
18.
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Increased Apolipoprotein Deposits in Early Atherosclerotic Lesions Distinguish
Symptomatic From Asymptomatic Patients
Moritz Wyler von Ballmoos, Denise Dubler, Martina Mirlacher, Gieri Cathomas, Jürgen Muser
and Barbara C. Biedermann
Arterioscler Thromb Vasc Biol. 2006;26:359-364; originally published online December 1,
2005;
doi: 10.1161/01.ATV.0000198250.91406.6d
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