Early Human Atherosclerosis
Accumulation of Lipid and Proteoglycans in Intimal Thickenings Followed
by Macrophage Infiltration
Yutaka Nakashima, Hiroshi Fujii, Shinji Sumiyoshi, Thomas N. Wight, Katsuo Sueishi
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Objective—The present study was designed to clarify the morphological features of early human atherosclerosis and to
determine whether specific extracellular matrix proteoglycans play a role in early atherogenesis.
Methods and Results—Step and serial sections were obtained from right coronary arteries with no or early atherosclerosis.
Atherosclerosis was classified into 4 grades according to the amount of lipid deposition. Coronary arteries with Grade
0 showed diffuse intimal thickening (DIT) with no lipid deposits. The extracellular matrix proteoglycans, biglycan and
decorin, were localized in the outer layer of DIT. Most cases of Grade 1 and Grade 2 exhibited fatty streaks with
extracellular lipids colocalizing with biglycan and decorin in the outer layer of the intima. As lipid grades increased,
macrophages increased in number and were present in the deeper layers. Most cases of Grade 3 exhibited pathologic
intimal thickening (PIT) with extracellular lipids underneath a layer of foam cell macrophages.
Conclusions—In early human coronary atherosclerosis, fatty streaks develop via extracellular deposition of lipids
associated with specific types of proteoglycans in the outer layer of preexisting DIT. As the amount of the lipid increases
in fatty streaks, macrophages infiltrate toward the deposited lipid to form PIT with foam cells. (Arterioscler Thromb
Vasc Biol. 2007;27:1159-1165.)
Key Words: early human atherosclerosis 䡲 diffuse intimal thickening 䡲 fatty streak 䡲 lipid retention 䡲 biglycan
L
ittle is known as to how early human atherosclerosis
develops. We previously reported that diffuse intimal
thickening (DIT) develops from an early age in human
arteries before atherosclerosis evolves.1 DIT, also known as
“nonatherosclerotic” intimal thickening,2 is a thickened intima mainly composed of smooth muscle cells (SMCs),
elastin, and proteoglycans, and devoid of lipid deposition. As
DIT is strongly expressed in atherosclerosis-prone arteries,
such as coronary arteries and abdominal aorta, we suggested
that DIT plays an important role in human atherogenesis. In
the classic pathological study, Holman et al showed that the
fatty streak, a nonraised sudanophilic lesion, is the earliest
lesion that appears in the aorta of children and adolescents
and some fatty streaks convert into the advanced raised lesion
in later life.3 This fact was also recently confirmed in the
coronary artery by McGill et al in The Pathobiological
Determinants of Atherosclerosis in Youth (PDAY) study.4
However, as microscopic examinations were not performed
in these studies, it is not clear how fatty streaks develop in
normal arteries and covert into advanced lesions. Based on
microscopic findings, Virmani et al defined the pathologic
intimal thickening (PIT) as a preatheromatous lesion that is
composed of extracellular lipid pools with an overlying layer
of SMCs and lipid-laden macrophages.2,5 These studies have
contributed to our understanding of the microscopic features
of human atherosclerosis before the stage of advanced lesions. Thus, PIT is thought to be an intermediate stage that
represents the link from early to advanced lesions,5 but the
nature of the early lesion and how the early lesion is
converted into PIT are yet to be clarified. Furthermore,
Williams and Tabas proposed the response-to-retention hypothesis in early atherogenesis in 1995, which states that
atherogenic lipoproteins are retained in the intima by binding
to extracellular proteoglycans.6 This hypothesis further states
that lipoprotein–proteoglycan complexes exhibit increased
susceptibility to oxidation and lead to uptake by macrophages
to form foam cells. Recent biochemical and molecular biological studies support this hypothesis.7 However, it is still
not clear whether this hypothesis is applicable to human
atherogenesis, partly because of the lack of morphological
evidence of early lesions documenting the presence and
location of these macromolecules.
See page 986
In the present study, we examined human coronary arteries, aiming to clarify the morphological features of the early
Original received June 16, 2006; final version accepted January 26, 2007.
From Pathophysiological and Experimental Pathology (Y.N., H.F., S.S., K.S.), Graduate School of Medical Sciences, Kyushu University, Japan; the
Division of Pathology (Y.N.), Fukuoka Red Cross Hospital, Fukuoka, Japan; and the Hope Heart Program at The Benaroya Research Institute and the
University of Washington (T.N.W.), Seattle.
Correspondence to Yutaka Nakashima, MD, PhD, Division of Pathology, Fukuoka Red Cross Hospital, 3-1-1 Ogusu, Minami-ku, Fukuoka 815-8555,
Japan. E-mail [email protected]
© 2007 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
1159
DOI: 10.1161/ATVBAHA.106.134080
1160
Arterioscler Thromb Vasc Biol.
May 2007
phases of atherosclerosis and the relationships between DIT,
fatty streaks, and PIT. Furthermore, we wished to determine
whether specific extracellular matrix (ECM) proteoglycans
such as biglycan and decorin are associated with lipid
deposits in the early lesion in support of the response-toretention hypothesis.
Methods
An expanded Methods section is available online at
http://atvb.ahajournals.org.
Autopsy Subjects and Coronary Arteries
A middle segment of the right coronary artery (RCA) together with
acute marginal branch was obtained from 38 Japanese autopsied
subjects who died between 7 and 49 years of age. The average of
serum total cholesterol and triglyceride levels were within normal
limits.
Definitions of DIT and Atherosclerotic Lesions
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In the present study, DIT was defined as a concentric intimal
thickening composed of SMCs, elastin, and proteoglycans and
devoid of lipid deposits.1,2 The fatty streak was defined as a
nonraised sudanophilic lesion.3 PIT was defined as a lesion with an
accumulation of extracellular lipids underneath a layer of lipid-laden
macrophages.2,5
Two-Dimensional Image of the Arterial
Cross Section
Step and serial cryostat sections were stained with elastica van
Gieson (EVG) and Sudan IV stains and immunostained with antiCD68 antibody. Intima, media, and lipids and macrophages existing
in the intima and media, were extracted from the digital images of
the stained sections (Figure 1a, 1b, 1c), and composed into a single
two-dimensional (2D) image (Figure 1d).
Reconstruction of a Three-Dimensional Image
A three-dimensional (3D) image of the artery was reconstructed from
a series of the 2D images (Figure 1e, 1f, 1g; supplemental Movie I,
available online at http://atvb.ahajournals.org). The ratio of the lipid
volume to the arterial wall (intima ⫹ media) volume was calculated.
Vertical Distribution of Lipids and Macrophages
The total lipid density (the ratio of the total lipid area to the total
arterial wall area) was calculated in 2D images. The arterial wall was
divided into 4 layers, ie, inner intima, outer intima, inner media, and
outer media (Figure 2a), to calculate the percentage of the lipid area
in the respective layer. The same measurements and calculations
were performed for macrophages.
Topographical Relationship Between Lipids and
Intimal Components
Cryostat sections were immunostained with antibodies
poprotein B (apoB), ␣-smooth muscle actin (␣-SMA),
biglycan (LF-121),8 decorin, monocyte chemoattractant
(MCP-1, F9),9 and oxidized phosphatidylcholine (ox-PC,
to apoli␣-elastin,
protein-1
DLH3).10
Results
Grading of Lipid Deposition and Spatial
Distributions of Lipids and Macrophages
According to the ratio of the lipid volume to the arterial wall
volume (⫻100) analyzed in 3D images, the grade of lipid
deposition in the whole arterial segment was classified into 4
grades: Grade 0, 0 (n⫽6); Grade 1, 0.01 to 0.50 (n⫽12);
Grade 2, 0.51 to 5.00 (n⫽13); Grade 3, 5.01 or more (n⫽7).
Lipid grades correlated positively with mean ages (ANOVA,
Figure 1. Construction of 2D and 3D images. a, A digital image
of intima and media (right) extracted from a EVG stained section
(left). I indicates intima; M, media. b, A digital image of lipids in
the intima and media (right) extracted from a Sudan IV stained
section (left). c, A digital image of macrophages in the intima
and media (right) extracted from an immunostained section with
anti-CD68 antibody (left). d, A 2D image composed of the digital
images of intima, media, lipids and macrophages. e, A 3D
image of the artery reconstructed from a series of 2D images. f
and g, The arterial wall was made translucent to observe the
distribution of lipids and macrophages, respectively. Lipid grade
in Grade 2, fatty streak. 39 years of age, female.
P⬍0.0001), but not with serum total cholesterol, triglyceride
levels, or smoking status (supplemental Table I). One of the
two cases with diabetes mellitus (27-year-old female) was in
Grade 1 and the other (49-year-old male) was in Grade 2. In
most cases of Grade 1 through Grade 3, lipids were deposited
eccentrically and more strongly in the proximal and branching portions than the distal portion (Figure 1f; supplemental
Movie I). These results demonstrate that this lipid deposition
is an early stage in human atherosclerosis. Macrophages were
sparsely distributed throughout the arterial wall in Grade 0
and Grade 1, and increased in Grade 2 and Grade 3 particularly around the deposited lipids (Figure 1g).
Vertical Distributions of Lipids and Macrophages
Figure 2b and 2c shows the total lipid density and the vertical
distribution of lipids in the arterial wall, respectively. No lipid
deposits were seen in Grade 0. Lipids occupied small areas of
the arterial wall in Grade 1. Most of the lipid existed in the
outer intima (hatched bar) and only a small proportion was
found in the inner intima (dotted bar) and inner media (open
bar). In Grade 2, the total lipid density was increased, and at
the same time, the proportion of the lipid area in the inner
intima was increased. This trend was more remarkable in
Grade 3 than Grade 2, although a large proportion of the
lipids was still present in the outer intima.
Nakashima et al
a
inner intima
outer intima
I
inner media
outer media
M
macrophage
lipid
b
d
30
8
6
20
%
%
4
10
2
0
c
0
e
100
100
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80
80
60
60
%
%
40
40
20
20
0
0
0
1
2
3
(6) (12) (13) (7)
grade
(n)
0
1
2
3
(6) (12) (13) (7)
Figure 2. Density and vertical distribution of lipids and macrophages in the arterial wall. a, Four layers of the arterial wall
divided as described in supplemental Methods section. I indicates intima; M, media. b, Total lipid density defined as the ratio
of total lipid area to total arterial wall (intima ⫹ media) area.
ANOVA, P⬍0.0001. c, The percentage of lipid area in the inner
intima, outer intima, inner media, and outer media (from top to
bottom) to total lipid area, which was averaged over the number
of cases in each grade. d, Total macrophage density defined as
the ratio of total macrophage area to total arterial wall area.
ANOVA, P⬍0.005. e, The percentage of macrophage area in the
inner intima, outer intima, inner media, and outer media (from
top to bottom) to total macrophage area, which was averaged
over the number of cases in each grade.
Figure 2d and 2e shows the total macrophage density and
the vertical distribution of macrophages in the arterial wall,
respectively. Although occupying only small areas, macrophages were seen even in Grade 0 and their distribution was
largely confined to the inner intima (dotted bar). The total
macrophage density and their distribution in Grade 1 were
almost the same as those of Grade 0. In Grade 2, the total
macrophage density was increased and so was the proportion
of the macrophage area in the outer intima (hatched bar). This
trend was greater in Grade 3 than Grade 2, although a large
proportion of the macrophages was still present in the inner
intima. A small proportion of macrophages was seen in the
outer media (cross-hatched bar) in Grade 0 through Grade 3.
The ratio of total macrophage area to total lipid area tended
to be larger in smokers than nonsmokers, although there was
no statistical significance (Mann–Whitney U test, P⫽0.075).
The ratio in 2 diabetic patients was not different from that in
nondiabetic patients of similar age.
Morphology of Early Human Atherosclerosis
1161
Microscopic Findings
Figure 3 shows representative micrographs of lipids and
macrophages. The arteries in Grade 0 exhibited DIT. There
were no lipid deposits, but a few macrophages in the
superficial layer of the thickened intima (Figure 3a, 3b, 3c).
All 12 cases in Grade 1 exhibited the mildest form of the fatty
streak. Mild lipid deposition was seen in the deep layer of the
intima, whereas macrophages showed no differences from
those of Grade 0 (Figure 3d, 3e, 3f). Ten of 13 cases in Grade
2 and 1 of 7 cases in Grade 3 also exhibited fatty streaks, but
lipids were more intensely and widely stained and macrophages were slightly increased in number and infiltrated
deeper than Grade 1 (Figure 3g through 3l). Most of the lipids
were localized in the outer layer of the intima but small
amounts were seen in the inner intima and inner media.
Higher magnification revealed that the lipids were localized
extracellularly and suggested that some lipids were associated
with elastin (supplemental Figure I). Even in cases with
greater amount of lipid deposits, the region affected by the
fatty streak was not raised as shown in Figure 1a and 1b.
Three of 13 cases in Grade 2 and 6 of 7 cases in Grade 3
exhibited PIT. Extracellular lipids accumulated in the outer
intima and were overlaid by a layer of lipid-laden macrophages (Figure 3m through 3r). Compared with the fatty
streak, extracellular lipids were more widely stained and
macrophages were more increased in number and more
deeply infiltrated in PIT. Foam cell macrophages were found
in 8 of 9 cases of PIT. In 7 cases (2 cases in Grade 2 and 5
cases in Grade 3), foam cells were present in and around the
interface between infiltrating macrophages and extracellularly deposited lipids (Figure 3p, 3q, 3r). One case in Grade
2 showed a small aggregate of foam cells in the superficial
layer of the intima. The region affected by the PIT lesion was
generally more thickened than the region without the lesion.
The correlation between lipid grades and atherosclerotic
lesions is summarized in supplemental Table I. All cases in
Grade 0 showed DIT. In Grade 1 through Grade 3, the fatty
streak and PIT were seen significantly in lower and higher
grades, respectively (Mann–Whitney U test, P⬍0.001).
Topographical Relationship Between Lipids and
Intimal Proteoglycans
Topographical relationships between extracellular lipids and
ECM proteoglycans were examined in 21 cases. First, the
distribution of biglycan and decorin was examined in the area
with no lipid deposits in 12 cases (Figure 4a). In 9 cases,
biglycan was concentrically and extracellularly localized
predominantly in the outer layer of DIT (Figure 4b, 4c).
Similar distribution was seen for decorin in 3 cases, but the
positive area in the intima was generally smaller than those of
biglycan (Figure 4d). No staining was observed in 3 cases for
biglycan and 9 cases for decorin. The average age of the
positive cases was older than the negative cases, although no
statistical significance was found (29.3⫾10.4 versus
15.7⫾8.3 for biglycan, 36.3⫾7.5 versus 22.4⫾10.5 for
decorin).
Second, correlations between the distribution of lipids and
that of biglycan and decorin were examined in 9 cases with
severe and/or diffuse extracellular lipid deposits in fatty
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Arterioscler Thromb Vasc Biol.
May 2007
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Figure 3. Representative histology of
deposited lipids and infiltrating macrophages in the arterial wall. a, b, and c,
Grade 0, DIT, 36 years of age, male. d,
e, and f, Grade 1, fatty streak, 32 years
of age, male. g, h, and i, Grade 2, fatty
streak, 39 years of age, female. j, k, and
l, Grade 2, fatty streak, 44 years of age,
male. The total lipid density was greater
in this case than the case of Figure g, h
and i. m, n, and o, Grade 3, PIT, 49
years of age, male. An accumulation of
lipid-laden (but not foamy) macrophages
was seen in the upper to middle layer of
the intima. p, q, and r, Grade 3, PIT with
foam cells, 29 years of age, male. Foam
cells with clear cytoplasm in EVG stained
section and abundant intracellular lipids
in Sudan IV stained section were accumulated in the upper to middle layers of
the intima. a, d, g, j, m, and p, EVG
stain. I indicates intima; M, media. b, e,
h, k, n, and q, Sudan IV stain. c, f, i, l, o,
and r, Immunostaining with anti-CD68
antibody. Arrowheads indicate internal
elastic lamina. Bars represent 100 ␮m.
DIT indicates diffuse intimal thickening;
PIT, pathologic intimal thickening.
streaks. In all 9 cases, the distribution of apoB coincided with
that of the Sudan IV positive area (supplemental Figure IIa,
IIb). Biglycan was colocalized with apoB in all 9 cases
(supplemental Figure IIc). The same finding was observed for
decorin in 6 cases (supplemental Figure IId), and no staining
in 3 cases.
Localizations of Oxidized Lipoproteins and MCP-1
Ox-PC was present extracellularly in the intima and tended to
be colocalized with apoB when relatively large amount of
lipids were deposited (supplemental Figure IIIa, IIIb). Ox-PC
was also seen intracellularly (supplemental Figure IIIa, IIIc).
MCP-1 was found in most of the macrophages and some
SMCs in the intima (supplemental Figure IIId).
Discussion
Initial Event of Human Atherosclerosis:
Extracellular Lipid Deposition in DIT and
Fatty Streaks
Only a few microscopic studies are available that examine the
earliest stage of human atherosclerosis, and the results are
inconsistent. For example, isolated macrophage foam cells
were believed to be the earliest sign of atherosclerosis,
whereas other studies suggested that intimal fibroplasia was a
first stage. In the present study, we used step and serial
sections of coronary arteries to detect subtle pathologic
changes, and found that the earliest stage of human coronary
atherosclerosis is the fatty streak that develops via extracellular deposition of apoB-containing lipids in the outer layer of
preexisting DIT. As the lesion progresses, lipids continue to
accumulate in the outer layer of the thickened intima of the
fatty streak without causing a remarkable change in intimal
thickness. Recently, extracellular lipid deposits in the intima
of human early lesions were also found by other
investigators.11,12
It is generally believed that extracellular lipids originate
from dead foam cell macrophages and accumulate to form a
lipid core in the advanced lesion. However, a few studies
have reported that lipids accumulate in the intima before
macrophages infiltrate in the early lesion. Napoli et al found
that lipids accumulated in the intima of human fetal aorta in
Nakashima et al
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Figure 4. Serial sections showing the localization of biglycan
and decorin in DIT. a, DIT with no lipid deposits. Sudan IV stain.
I indicates intima; M, media. b, Immunostaining with antibiglycan antibody. c, Higher magnification of the outer layer of
DIT in b, showing extracellular localization of biglycan. d, Immunostaining with anti-decorin antibody. 36 years of age, male.
Arrowheads indicate internal elastic lamina. Bars represent
100 ␮m (a, b, d) and 20 ␮m (c).
the absence of macrophages.13 Investigating the human aorta
of young and middle-aged adults, Guyton and Klemp suggested that an early lipid core arises from lipids accumulating
gradually in the ECM of the deep intima.14 In the present
study, we confirmed that the initial lipid deposition occurred
independently of foam cell death, as no such cells existed
around the deposited lipids. Accumulation of lipids prior to
the macrophage infiltration is also reported in animal models,15 suggesting that the same molecular mechanism occurs
both in experimental animals and humans. However, it is
noteworthy that the environment where lipids deposit is
considerably different. In experimental animals, lipids deposit in the narrow subendothelial space that consists almost
exclusively of ECM and contains few SMCs.15 However, in
humans, the lipid deposits initially in the deep layer of the
thickened intima of DIT which contains abundant ECM and
SMCs.1,2
Macrophage Infiltration and Formation of PIT
With Foam Cells
The present study suggests that macrophages are stimulated
to accumulate in the fatty streak, infiltrate toward the deposited lipids, and subsequently form the PIT, as extracellular
lipids increase in amount. According to in vitro studies,
chemotactic movements of macrophages are induced via
modification of low-density lipoprotein (LDL) and stimulation of MCP-1 production by cells in the lesion.16 These
mechanisms are expected to occur in vivo as well, because
the presence of oxidized lipoproteins and MCP-1 was revealed in the intima in the present study. The existence of
oxidized and enzymatically-modified LDL and MCP-1 is also
demonstrated in the human arterial wall in other studies.9,11,12
Cigarette smoking may enhance macrophage infiltration in
the intima as shown in the present study. This enhancement
may be related to the modification of LDL and/or inflamma-
Morphology of Early Human Atherosclerosis
1163
tory factors such as adhesion molecules by toxic substances
in the smoke.
The present study also suggests that the transformation
from macrophages to foam cells is achieved by phagocytizing
deposited lipids, because foam cells were primarily formed in
and around the interface between infiltrating macrophages
and extracellular lipids. Phagocytizing lipid by macrophages
is believed to be an essential mechanism to form foam cells
in animal models as well, but microscopic features of the
lesions containing foam cells are considerably different from
those of humans. In animal models, foam cell macrophages
appear in the initial stage of atherosclerosis and occupy the
whole thickness of the intima as a predominant component of
the lesion.17 As shown in the present study, foam cells are
formed in human PIT lesions subsequently to the deposition
of a certain amount of extracellular lipid, together with SMCs
and proteoglycans.2,5
Role of Proteoglycans in Extracellular
Lipid Deposition
A number of biochemical and molecular biological studies
suggest that the lipid binding capacity of proteoglycans
contributes to retaining atherogenic lipoproteins in the intima.
However, morphological evidence and specific location of
the components involved in proteoglycan-lipoprotein accumulation is lacking. The present study illustrates that biglycan
occurs extracellularly in the outer layer of DIT in the exact
location as the early distribution of lipids (note the similarity
of the prelesional distribution of biglycan in DIT in Figure 4b
and the early distribution of lipids in the fatty streak in Figure
3h). However, it is of interest that lipids deposit eccentrically,
whereas biglycan is localized concentrically. It is believed
that structural changes in the glycosaminoglycan (GAG)
chains on proteoglycans are the initial proatherogenic step
that leads to increase binding properties of proteoglycans for
atherogenic lipoproteins.18 For example, proteoglycans produced by transforming growth factor (TGF)-␤1–treated cultured SMCs show longer GAG chains and greater binding
affinity to LDL than control SMCs.19 Interestingly, patchy
distribution of TGF-␤1 is found in DIT.20 It is also noteworthy that mechanical strain, which is thought to be unevenly
distributed in the arterial wall, upregulates biglycan mRNA
expression in cultured SMCs.21 These results suggest that
regional differences in the quality and quantity of biglycan
exist in the intima and possibly lead to regional differences in
the amount of lipid deposition. Two other mechanisms that
may cause regional differences in the lipid distribution are
uneven plasma lipoprotein concentration and the permeability
of the arterial wall. Deng et al reported that luminal surface
concentration of LDL was increased in areas where wall shear
stress was low and suggested that increased surface LDL
concentration results in an increased lipid infiltration rate into
the intima.22 However, the relationship between permeability
of the arterial wall and susceptibility of atherosclerosis is
debatable. The permeability to LDL was greater in the
atherosclerosis-susceptible areas than atherosclerosis-resistant areas of the rabbit aorta,23 but opposite results were
obtained in white Carneau pigeon aorta.24
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Correlations between the distribution of lipids and proteoglycans have been investigated in early and advanced atherosclerotic lesions.25–27 A consistent finding, including that of
the present study, is the colocalization of biglycan and
apolipoproteins.25,26 It is also noteworthy that oxidized LDL
is capable of stimulating biglycan expression by SMCs and
enhancing the interaction of this proteoglycan with lipoproteins.28 It is tempting to speculate that the accumulation of
biglycan in specific regions of the early lesions may result
from the presence of the lipoproteins associated with the
SMCs. These molecular interactions may result in a vicious
cycle of atherosclerosis. We found that decorin appears to
colocalize with lipid in some instances but much less consistently than biglycan. Versican is another important extracellular proteoglycan in human atherogenesis, as it accumulates
in human atherosclerotic lesions,25,27 but not in mouse models.26 The role of versican may be different from that of
biglycan and decorin, because its distribution is different
from biglycan and decorin.25,29 In our preliminary study,
versican was predominantly localized in the inner layer of
DIT and fatty streaks. Diffuse distribution of versican across
the intima was also seen in some cases. In advanced human
lesions, versican is present at the plaque thrombus interface,
suggesting a possible role in thrombosis.27
2.
3.
4.
5.
6.
7.
8.
9.
10.
Summary
The present study supports the response-to-retention hypothesis as being involved in the early phases of human coronary
atherosclerosis. Furthermore, this study highlights the importance of the DIT and the fatty streak as a reservoir for lipid
retention and identifies a family of ECM molecules that may
be involved in lipid retention, contributing to the early phases
of lesion formation before the stage of the PIT. Although only
coronary arteries were targeted in the present study, the same
mechanisms are expected to occur in other atherosclerosisprone arteries, such as abdominal aorta, because welldeveloped DIT is present in their intima as well.1
Acknowledgments
We thank Morishige Takeshita (Kyushu Medical Center, Fukuoka,
Japan) and Ichiro Yamamoto (Hamanomachi Hospital, Fukuoka,
Japan) for kindly providing case materials, and Larry W. Fisher
(Craniofacial and Skeletal Diseases Branch, National Institute of
Dental and Craniofacial Research, National Institutes of Health,
Bethesda, MD), Hiroyuki Itabe (Laboratory of Biological Chemistry,
School of Pharmaceutical Sciences, Showa University, Tokyo,
Japan), and Motohiro Takeya (Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan) for generously providing antibodies to biglycan,
ox-PC, and MCP-1, respectively.
11.
12.
13.
14.
15.
16.
17.
Sources of Funding
This work was supported in part by a research grant from the
National Institutes of Health (HL 18645) (to T.N.W.).
18.
Disclosures
19.
References
20.
None.
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Early Human Atherosclerosis: Accumulation of Lipid and Proteoglycans in Intimal
Thickenings Followed by Macrophage Infiltration
Yutaka Nakashima, Hiroshi Fujii, Shinji Sumiyoshi, Thomas N. Wight and Katsuo Sueishi
Arterioscler Thromb Vasc Biol. 2007;27:1159-1165; originally published online February 15,
2007;
doi: 10.1161/ATVBAHA.106.134080
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Supplemental Data for Nakashima et al.
Early Human Atherosclerosis: Accumulation of Lipid and Proteoglycans
in Intimal Thickenings Followed by Macrophage Infiltration
Methods
Autopsy subjects and coronary arteries
A middle segment of the right coronary artery (RCA) together with acute marginal branch
was obtained from 38 Japanese autopsied subjects who died between 7 and 49 years of age
(32.3 ± 11.2 years, 25 males and 13 females). The arteries with no or early stage of
atherosclerosis were examined in this study.
The autopsy subjects died of diseases, such as
malignancies and liver cirrhosis, but not coronary heart diseases. The average of serum total
cholesterol and triglyceride levels were within normal limits (150.0 ± 41.1 mg/dl, n=38 and 120.6
± 58.0 mg/dl, n=34, respectively).
Serum triglyceride levels were unknown in 4 cases.
Diabetes mellitus was found in two cases. Twenty subjects were smokers and 16 were
nonsmokers. Smoking status was unknown in 2 cases. The study was approved by the ethics
committee of the Department of Pathology, Kyushu University and performed in accordance with
the guidelines.
Definitions of diffuse intimal thickening (DIT) and atherosclerotic lesions
In the present study, DIT was defined as a concentric intimal thickening composed of
smooth muscle cells (SMCs), elastin and proteoglycans and devoid of lipid deposits.1, 2
According to the definition of Holman et al.,3 the fatty streak was defined as a non-raised
sudanophilic lesion. Pathologic intimal thickening (PIT) was defined according to the definition
of Virmani et al.2, 4 as a lesion with an accumulation of extracellular lipids underneath a layer of
lipid-laden macrophages.
Antibodies
Rabbit polyclonal antisera for human biglycan (LF-121) 5 were generously provided by Larry
W. Fisher, Craniofacial and Skeletal Disease Branch, National Institute of Dental and
Craniofacial Research, National Institutes of Health, Bethesda, MD and used at a 1/500 dilution.
Mouse monoclonal antibodies for monocyte chemoattractant protein-1 (MCP-1, F9) 6 and
oxidized phosphatidylcholine (ox-PC, DLH3) 7 that specifically recognize oxidized lipoproteins
including oxidized low density lipoprotein (ox-LDL) were generously provided by Motohiro
Takeya, Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto
University and Hiroyuki Itabe, Laboratory of Biological Chemistry, School of Pharmaceutical
Sciences, Showa University and used at 1/7 and 1/3200 dilutions, respectively.
Mouse
monoclonal antibodies for anti-human CD68, α-smooth muscle actin (α-SMA) and MCP-1 were
purchased from Dako (Glostrup, Denmark), Sigma (St. Louis, MO) and R & D systems
(Minneapolis, MN) and used at 1/100, 1/1000 and 1/50 dilutions, respectively.
Rabbit antisera
for human α-elastin and goat antisera for human decorin and apolipoprotein B (apoB) were
purchased from Biogenesis (Poole, England), R & D systems (Minneapolis, MN) and Rockland
(Gilbertsville, PA) and used at 1/100, 1/20 and 1/400 dilutions, respectively.
Two-dimensional image of the arterial cross section
Three serial cryostat sections of 10 micrometers thick were cut from the artery and grouped
in one unit. Twenty-nine to 50 units were obtained every 200 or 250 micrometers, which made
a total length ranging from 6.0 to 12.3 mm.
Two sections in each unit were stained with elastica
van Gieson (EVG) and Sudan IV stains to identify the arterial intima, media, and lipids,
respectively.
macrophages.
The other section was immunostained with anti-CD68 antibody to identify
Digital images of the stained sections were captured with a charge-coupled
device (CCD) camera.
Intima, media, and lipids and macrophages existing in the intima and
media, were extracted from these digital images using digital imaging software (Adobe
Photoshop, Adobe Systems Inc.) (Figure 1a, 1b, 1c), and composed into a single two-dimensional
(2D) image (Figure 1d).
Reconstruction of a three-dimensional image
A three-dimensional (3D) image of the artery was reconstructed from a series of the 2D
images using 3D reconstruction software (TRI, RATOC System Engineering, Tokyo, Japan) to
observe the spatial distribution of lipids and macrophages (Figure 1e, 1f, 1g, supplemental Movie
I), and to calculate the ratio of the lipid volume to the arterial wall (intima + media) volume.
Vertical distribution of lipids and macrophages
A 2D image with the most severe lipid deposits was selected from each case.
with a branching site was excluded.
The image
The total lipid density, which was defined as the ratio of
the total lipid area to the total arterial wall (intima + media) area, was calculated using digital
imaging software.
Then, the arterial wall was divided into four layers, i.e., inner intima, outer
intima, inner media and outer media, by two lines drawn circumferentially at the middle of the
intima and media, respectively (Figure 2a), to calculate the percentage of the lipid area in the
respective layer.
The same measurements and calculations were performed for macrophages.
The ratio of total macrophage area to total lipid area was calculated to determine the effect of
smoking and diabetes mellitus on macrophage infiltration.
Topographic relationship between lipids and intimal components
In 21 cases (31.9 ± 11.6 years of age, 14 males and 7 females), seven cryosections of 4
micrometers thick were obtained from the adjacent portion of RCA.
with EVG and Sudan IV stains.
Two sections were stained
The other five sections were immunostained with antibodies to
apoB, α-SMA, α-elastin, biglycan and decorin.
The distributions of ox-PC and MCP-1 were
also investigated by immunohistochemistry with two other cryosections.
Statistical analysis
Results were given in mean ± SD, and analyzed by ANOVA, Student’s t-test, and
Mann-Whitney’s U test.
P<0.05 was considered significant.
References
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Virmani R. Characterization of intimal changes in coronary artery specimens with MR
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Fisher LW, Stubbs JT 3rd, Young MF. Antisera and cDNA probes to human and certain
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Itabe H, Takeshima E, Iwasaki H, Kimura J, Yoshida Y, Imanaka T, Takano T. A
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Table
Table I.
Grading of lipid deposition and correlations of the grade with the atherosclerotic lesions and clinical data.
ratio of lipid volume to
total
arterial wall volume*
Grade
atherosclerotic
age
cholesterol
triglyceride
smoking
lesion
(years)
(mg/dl)
(mg/dl)
status¶
6
DIT (6) §
18.2 ±11.0║
175.2 ± 57.9 (6)
148.8 ± 76.9 (6)
S (2) NS (4)
FS (12)
29.7 ± 9.3
142.3 ± 30.0 (12)
114.2 ± 60.8 (11)
S (7) NS (4) UK (1)
†
(x100)
‡
n
0
0
1
0.01 - 0.50
12
2
0.51 - 5.00
13
FS (10)
PIT (3)
35.2 ± 7.0
142.4 ± 44.2 (13)
93.5 ± 37.3 (11)
3
5.01 -
7
FS (1)
PIT (6)
43.4 ± 6.8
155.7 ± 33.9 (7)
153.7 ± 45.9 (6)
*
arterial wall volume = intimal volume + medial volume
†
n: number of cases in each grade
‡
DIT: diffuse intimal thickening,
§
( ): number of cases
FS: fatty streak,
║ mean ± SD
¶
S: smoker,
NS: nonsmoker,
UK: unknown
PIT: pathologic intimal thickening
S (5) NS (8)
S (6) NS (0) UK (1)
Movie
Movie I. (Please see the supplemental movie.) Spatial distribution of lipids in the arterial
wall.
The movie is an animation of the model shown in Figure 1f.
Lipids deposited on the
epicardial surface and not on the myocardial surface of the coronary artery in this case.
39 years
of age, female.
Figures
Figure I.
Serial sections showing the extracellular localization of lipids.
Lipids deposited in
the extracellular space of the deep intima (a) shown as an unstained area in a section
immunostained for smooth muscle cells (b).
Some lipids were aggregated to demonstrate wavy
appearance (a) similar to that of elastic fibers (c), suggesting the association of lipids with elastin.
(a): Sudan IV stain.
(b): Immunostaining with anti-α-SMA antibody.
with anti-α-elastin antibody.
lamina. Bars represent 20 µm.
44 years of age, male.
(c): Immunostaining
Arrowheads indicate internal elastic
Figure II.
Serial sections showing the correlations between the localization of apoB and that
of biglycan and decorin in the fatty streak.
the Sudan IV positive area (a).
(a): Sudan IV stain.
The distribution of apoB (b) coincided with that of
Biglycan (c) and decorin (d) were colocalized with apoB (b).
I: intima, M: media.
(b): Immunostaining with anti-apoB antibody.
(c):
Immunostaining with anti-biglycan antibody. (d): Immunostaining with anti-decorin antibody.
43 years of age, male.
Arrowheads indicate internal elastic lamina.
Bars represent 100 µm.
Figure III.
Serial sections showing the localization of ox-PC and MCP-1.
Ox-PC was
present extracellularly in the outer layer of the intima (a) and colocalized with apoB (b).
also existed in the cells (a).
Ox-PC
Round cells in the upper layer were macrophages, as shown in
figure c, and polygonal and spindle cells in the deeper layer were SMCs. MCP-1 was found in
most of the macrophages infiltrating in the upper layer of the intima and some SMCs (arrows) in
the deeper layer (d).
(a, b, c, d): Immunostaining with anti-ox-PC (a), anti-apoB (b), anti-CD68
(c) and anti-MCP-1 (d) antibodies, respectively.
The magnification in figure d is twice as high
as that in other figures so that the positive cells are clearly shown.
Arrowheads indicate internal elastic lamina.
33 years of age, male.
Bars represent 50 µm (a, b, c) and 20 µm (d).