Americani Jouirnal of Pathology, Vol. 146, No.
1, January 1995
Distribution of Unesterified
Cholesterol-Containing Particles in Human
Atherosclerotic Lesions
Sara Sarig, Wulf H. Utian, Leon A. Sheean,
and George 1. Gorodeski
From the Department of Reproductive Biology, Case Western
Reserve University School ofMedicine, and the Department
of Obstetrics and Gynecology, University MacDonald
Women 's Hospital, University Hospitals of Cleveland,
Cleveland, Ohio
The objective of the study was to characterize un-
esterified cholesterol particles in human aorta
and to correlate the findings with the severity of
aortic atherosclerosis. Human tissues were pro-
cessed under conditions that preserve deposits of
unesterified cholesterol agglomerates. Filipin-
fluorescence was determined by using a novel
triple bandpassfilter. The pattern of unesterified
cholesterol deposits was age related and corre-
lated with the severity of atherosclerosis. We
found three types of deposits: 1), smaU spheru-
lites (3 to 5 u), which were depicted in both the
media and intima in individuals as early as age
16, and which, in more advanced ages, showed an
increase in density and a tendency to aggregate
extracelularly throughout the intima in clusters;
2), elongated structures (10 to 30.u in the middle
zone of the intima), the density of which was di-
rectly related to the severity of atherosclerosis;
and 3), large (100 u), irregular deposits found
mainly in the core of atherosclerotic plaques. The
medium size deposits, compared with those
found in the core of atherosclerotic plaques, re-
tain their overaU size (10 to 30 ), uniformity
(oval elongated), and localization (middle zone
of the intima). On the basis of these observations
we hypothesize cholesterol deposition in two
stages of aggregation 1), early degradation of
infiltrating low density lipoprotein particles
forming unesterified cholesterol-rich vesicles in
the vessel wall, folowed by aggregation to
spherulites in the lowerpart ofthe intima; and 2),
more massive agglomeration ofparticles contain-
Copyright C) American Society for Investigative Pathology
ing unesterified cholesterol and calcium phos-
phate in the midzone of the intima. Because in the
second stage of aggregation the transition of cho-
lesterol to the solid state has already occurred, it
is irreversible. (Am JPathol 1995, 146:139-147)
Atherosclerotic cardiovascular disease is the leading
cause of morbidity and mortality in men and women.1
The prevailing theory of atheroma formation indicates
accumulation and deposition of lipids, including cho-
lesterol, in extracellular tissues of arterial walls, but
little is known about the actual mechanism of cho-
lesterol agglomeration. In particular, little is known
about the role of unesterified cholesterol in athero-
genesis, and most of our knowledge is derived from
studies that focused on the esterified form of choles-
terol. Unesterified cholesterol constitutes the major
part of the total cholesterol mass of degraded low
density lipoprotein (LDL) and is therefore likely to play
a role in lipid accumulation in peripheral tissues. Pre-
vious studies have failed to depict the unesterified
cholesterol in arterial walls in situ, partly because of
inappropriate methodology to detect lipids. Routine
histological techniques use organic solvents to fix tis-
sues, but these solvents solubilize the unesterified
cholesterol.
The objective of this study was to use the fluores-
cent agent filipin to detect unesterified cholesterol
aggregates in human arteries. The fluorescent poly-
ene antibiotic filipin binds specifically to 3-0-
hydrosterols2'3 and was used to assay unesterified
cholesterol deposits in experimental animal lesions4
and in atherosclerotic human tissues.5'6 Our objective
was to use this probe to localize and characterize
unesterified cholesterol deposits in human arteries
obtained at autopsy and to correlate the intensity of
the fluorescent pattern with the severity of the ath-
erosclerotic lesion.
Accepted for publication August 19, 1994.
Address reprint requests to Dr. George I. Gorodeski, University
MacDonald Women's Hospital, University Hospitals of Cleveland,
2074 Abington Road, Cleveland, OH 44106.
139
 140 Sarig et al
AJPJanuary 1995, Vol. 146, No. 1
Extracellular filipin-positive particles represent a
previously unrecognized structure in which unesteri-
fied cholesterol accumulates within atherosclerotic
lesions.4 Previous studies described the presence of
filipin-positive particles in rat, rabbit, and monkey
atherosclerotic lesions induced by cholesterol feed-
ing. It was found that early accumulation of filipin
fluorescent-positive material occurs in the intima, es-
pecially against the elastica interna.7 In human ath-
erosclerotic lesions, filipin staining occurred in asso-
ciation with three extracellular structures, spherical
particles, elongated crystals, and granular calcium
deposits, neither of which stained by red oil-O. These
results indicated that accumulation of unesterified
and esterified cholesterol may occur in separate ex-
tracellular regions of the arterial wall.5
In earlier studies, calcium phosphate-cholesterol
agglomerates containing unesterified cholesterol
were investigated both in vitro and in segments of
human atherosclerotic tissues,6 and the existence of
apatite-cholesterol agglomerates in human athero-
sclerotic lesions was verified in a study with five di-
verse electron microscope techniques.8 Cholesterol-
hydroxyapatite agglomerates, produced in vitro,
which simulate the calcium phosphate-cholesterol
deposits in lesions9 stained with filipin-emitted
fluorescence in a manner very similar to the fluores-
cence motif obtained from the filipin staining of the
tissue.6 Those studies were focused on the com-
parison between natural and artificial cholesterol-
hydroxyapatite agglomerates, and the experimental
procedure adapted in those studies6 included immer-
sion of the aortic tissue in ethanol for 30 minutes to
eliminate all of the filipin staining except that associ-
ated with calcium deposits, ie, all of the free unest-
erified cholesterol accumulations. In the present
study, to prevent unintentional dissolution of unes-
terified cholesterol deposits in the tissue, the tissue
sections prepared for filipin staining were not treated
with any organic solvent. The problem of differenti-
ating the filipin-cholesterol fluorescence from the
autofluorescence of elastin and cellular inclusions
has been addressed4 and resolved by using con-
secutively two separate filters for the same field of
view and eliminating, by visual inspection, the
autofluorescent signal that was seen with blue light.
In a previous study carried out in our laboratory,10 the
fluorescence patterns obtained from filipin-stained
human atherosclerotic lesion were studied by using a
blue standard and a green dual filter. Though it was
possible to identify the filipin-cholesterol complex by
comparison between the blue light and the green light
pictures, the procedure was strained. Subsequently,
we described an improved methodology to specify
the filipin fluorescence by using a triple band pass
filter. The use of the triple band pass filter resolved the
difficulty of differentiating between filipin-cholesterol
and tissue endogenous fluorescence, registering the
integrated fluorescence pattern on a single photomi-
crograph in different colors. The background tissue
appears gray and the small circular particles con-
taining unesterified cholesterol (identified by its green
fluorescence when stained with filipin10) are pink,
whereas the large elongated agglomerates, evidently
composed of cholesterol and calcium phosphate,
stain brilliant white.10
We used this novel microscopic method to survey
a number of human arteries and to observe the dis-
tribution of unesterified cholesterol-containing par-
ticles in human arteries of donors within a wide range
of ages and of severity of the atherosclerotic lesions.
Materials and Methods
Patients
Human arteries were obtained at autopsy from pa-
tients, males and females aged from 16 to 83 years,
who died from diverse causes. Histological diag-
noses and the causes of death were obtained from
the Department of Pathology at University Hospitals
of Cleveland.
Tissue Preparation
After adventitial tissues were removed, arteries were
opened longitudinally and segments of 1 to 2 inches
were wrapped in aluminum foil and snap frozen in
liquid nitrogen. Cross sections of approximately one
quarter of an inch were cut and mounted on cork-
boards and kept frozen at -80 C until subjected to thin
section cutting with a steel knife in a Reichert Junge
International Equipment Co. apparatus at -19 C. The
sections (14 p thick) were mounted on microscopic
slides. Sections for red oil-O staining (which demon-
strates general lipids and esterified cholesterol) were
processed with red oil-O solution in 99% isopropanol,
stained for 45 minutes, counterstained for 30 seconds
in Harris hematoxylin, rinsed in tap water, rinsed in
0.05% lithium carbonate for 15 seconds, rinsed in tap
water again, mounted in glycerin jelly, and kept under
subslides. Sections intended for filipin staining were
stored at -80 C until assayed. The filipin stain solution
was prepared by dissolving 2.5 mg of solid filipin
complex (F9765, Sigma Chemical Co., St. Louis, MO)
in 1 ml of dimethylformamide and adding the solution
to 50 ml of diphosphate buffer saline. Before viewing

with a light fluorescent microscope, the filipin solution
was layered above a frozen unfixed section and in-
cubated for 20 minutes.
Microscope and Filters
A Zeiss fluorescent Axiophot photomicroscope was
used. The UV excitation was carried out with a super
pressure mercury lamp, HBO 100 W/2 at 365 nm,
and the viewing was performed with a triple band
pass 4',6-dianidino-2-phenylindole dihydrochloride/
fluorescein isothiocyanate/Texas red filter set-up with
excitation wavelengths 405, 490, and 570 nm and
emission wavelengths 465, 530, and 640 nm (61002;
Chroma Technology Corp., Brattleboro VT).
Results
Table 1 lists the data on the patients whose arteries
were examined for deposits of unesterified choles-
terol with the fluorescent filipin probe. Sections of the
arteries, untreated with organic solvents and pre-
served at -80 C, were incubated with filipin and
viewed with a fluorescence microscope with the triple
band pass filter. This filter allows us to distinguish on
a single photomicrograph between the endogenous
fluorescence of the arterial tissue and the fluores-
cence of the unesterified cholesterol-filipin complex,
by virtue of color differences.
The segment of aorta shown in Figure 1A is from a
16-year-old male (Table 1, patient 1), and it depicts a
nonthickened intima (left upper part) and an expanse
of media with gaps between the tissue fibers. A few
separate small pink dots, approximately 5 p in size,
are dispersed in the media (arrows). Figure 1 B is an
enlargement of one such dot at magnification x200.
Figure 2 is a section of aorta obtained from a 27-
year-old woman (Table 1, patient 4). It shows a mildly
thickened but intact intima (upper part) and multiple
pink dots sprinkled within the intima. The latter appear
to be composed of chains of two to five dots arranged
horizontally, ie, in parallel to the surface of the tissue
Table 1. Clinical Data and Experimental MatenIal
Patient
No. Artery Cause of Death Sex Age
1 Aorta Hydrocephalus M 16
2 Aorta Viral encephalitis F 26
3 Aorta Hodgkin's disease F 27
4 Aorta Respiratory arrest F 27
5 Aorta Cystic fibrosis M 28
6 Aorta CAD, diabetes mellitus F 45
7 Aorta CAD M 66
8 Aorta Complicated aortic lesion M 83
CAD, coronary artery heart disease.
Unesterified Cholesterol in Human Aorta 141
AJPJanuary 1995, Vol. 146, No. 1
and in spaces between cells. In addition to the dis-
persed small pink dots, there are also several brightly
stained particles, approximately 20 p,, located in a
rather discrete area close to the elastic boundary be-
tween the intima and the media. A few small pink
dots can also be seen in the media. A similar pattern
of filipin staining was seen in two additional cases
(Table 1, patients 2 and 3, not shown).
Figure 3A is a section of aorta of a 28-year-old man
(Table 1, patient 5) with a more thickened and dam-
aged intima. Dispersed throughout the intima, but
particularly in the lower and upper zones, are numer-
ous small circular pink particles of a size of 3 to 10 p.
They are also arranged in small chains and/or clusters
that appear to be extracellular and lie in parallel to
the surface of the tissue. In the middle zone of the
intima, there are elongated white, strongly fluorescent
particles forming a trail. These particles vary in size
(approximately 20 p).
The five donors (Table 1, patients 1 to 5) had no
cardiovascular disease.
The aortic section in Figure 3B is of a 45-year-old
woman who died of atherosclerotic cardiovascular
disease (Table 1, patient 6). Figure 3B was used in our
former study of filipin staining in a different context10
to demonstrate the improved sensitivity of the novel
filipin staining technique and to compare it with the
use of green and blue filters. In the present study, it
serves to exhibit the spatial distribution of the filipin-
positive particles. As is shown in Figure 3B, filipin
staining can discern three types of structures 1), small
spherical particles at the base of the thickened intima
(approximately 5 to 10 Ip); 2), elongated, intensely
white large particles in the middle zone (approxi-
mately 20 to 80 p); and 3), pink, medium-sized par-
ticles (approximately 10 to 30 p) in the upper part of
the intima.
Figure 4A is a section of aorta of a 66-year-old man
with diffuse atherosclerotic cardiovascular disease
(Table 1, patient 7), showing a subintimal core lesion.
The upper part of the intima is not disrupted but, as
may be deduced from the gap in the right-hand side
of the figure and the remnants of the tissue above the
gap, the core of the lesion had developed under the
endothelium and pushed it into the arterial lumen. In
this section, as in the former ones (Figures 1 to 3),
filipin stained three types of structures, but the relative
distribution of the structures in this case differed
markedly compared with the former cases. Small pink
dots, (approximately 5 p) can be seen throughout the
intima (triangles), including in the core lesion. Most
abundant were large, filipin-positive agglomerates,
which stained brilliant white (approximately 20 to 60
p). These were found mainly in the core lesion (arrow)
142 Sarig et al
AJPJanuary 1995, Vol. 146, No. 1

Figure 1. Filipini staining of unesterified cholesterol aggregates: cross section of aorta fronm a 16-vear-old maoi (Table 1). 7he procedurefJirfilipin
stainitng and fluorescence microscopy are described in Materials and Methods. A: tpper part, nornmal inititna. Media is shown w'ith gaps between
tissue fibers. Several small pink dots (arrous) are dispersed in the media. These dots represent spherical cholesterol-containing particles. Jlagnifi-
cation, x 100. B: Same as (A), magnification X200. Note the pink stained dot. Bar = 1(0 0.

but also outside of the core lesion itself. In addition,
elongated structures (approximately 20 to 40 p) were
also stained brilliant white with filipin and can be seen
in the middle zone of the intima (square). In contrast
to the former, however, the elongated particles ap-
pear to be discretely extracellular and to lie parallel to
the surface of the endothelium.
Figure 4B is a section of an aorta of an 83-year-old
man who died of diffuse atherosclerotic cardiovas-
cular disease (Table 1, patient 8), showing a complex
 PE9duy;>.S^*\-etzNFW:;4~_lkjw, Ah;._
Unesterified Cholesterol in Human Aorta 143
AjPJanuary 1995, Vol. 146, No. ]

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A. ~ ~ ~ ~ ~~A

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Figure 2. Filipin staining of lnesterified cholesterol aggregates: cross section of aorta from a 27-year-old woman (Table 1). Note a thickened bht
intact intima and multiple pink dots in the loner part of the intima. Some dots aggregate in short chains (arrow). Several pink dots can also be
seen in the media. Bar = 100 y.

atherosclerotic deposit with thickened and damaged
intima. Despite its complex appearance, however, the
filipin staining had the same general pattern as in Fig-
ure 4A. As can be seen in Figure 4B, in the area that
corresponds to the middle zone of the intima and that
lies above the core of the lesion, filipin stained with
brilliant white an abundance of particles that were ap-
proximately 10 to 100 p. Some of these still retained
their elongated shape (arrow), whereas others were
more rounded. In the core of the lesion, filipin stained
diverse elements, some of which were large, irregular
agglomerates that stained pink-white (triangles),
whereas others were smaller and more condensed
structures that stained brilliant white. In addition, the
tn\*K_.t, .uXS
144 Sarig et al
AJPJanuary 1995, Vol. 146, No. 1
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t
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Figure 3. Filipin staining of unesterified cholesterol aggregates. A:
Cross section of aorta from a 28-year-old man (Table 1). The intima
is thickened and damaged; areas in the upper part of the intima are
discotntinuous and disconnected. Smallpink dots are dispersed in the
lower and the upper zones of the intima, and large elongated, bril-
liant uhite fluorescent particles form a chain in the middle zone. B:
Cross section of aorta from a 45-year-old woman (Table 1). Note a
trail of large, elongated, strongly fluorescent particles in the middle
zone (arrou), small pink dots and diffuse fluorescence in the lower
zone, and medium size pink particles in the upper zone of the in-
tima. Bar = 100 }.
small circular pink dots were seen through the entire
section in considerable density. Overall, compared
with less severe cases of atherosclerosis (eg, Figures
3A, B and 4A), in the case presented in Figure 4B
there is more diversity, intermingling, and superim-
position of the different kinds of filipin-stained par-
ticles within the intima.
Discussion
The survey of human aortas obtained from patients of
ages 16 to 83 shows a spectrum of unesterified cho-
lesterol deposits, starting from the few isolated, small
particles (approximately 3 to 5 P) to dense agglom-
erates (tens of microns). The detection of diverse in-
termediate patterns suggests that the modified mi-
croscopic procedure used in the present study can
become a functional tool in the study of mechanisms
of atherosclerotic lesion formation.
Figure 4. Filipin staining of unesterified cholesterol aggregates. A:
Cross section of aorta from a 66-year-old man (Table 1). A siubinti-
mal core lesion is shown in the center of the figure. Small dots (tri-
angles) surrounding the core can be seen also inside the core. In ad-
dition to the small dots, two otherfilipin-positive stained particles are
evident: large particles of diverse sizes and shapes (arrou), seen
mainly inside the core itself and medium sized elongated particles,
seen in the middle zone of the intima (squares). B: Cross sectionl of
aorta from an 83-year-old man (Table 1). Note a subintimal core le-
sion in the center of the figure. Shown are the three types offilipin-
positive particles, as in (A). Most abundant are the medium sized
particles in the middle zone of the intima (arrow! points to al elon-
gated particle). In the core of the lesion can be seen large agglomer-
ates (triangles). Also seen are pink dots, scattered throu.ghouit the in-
tima. Bar= 100 .
Despite the diversity of the filipin-stained particles,
we have found a pattern of unesterified cholesterol
deposits in the human aorta that was age related and
correlated with the severity of atherosclerosis in the
vessel. We have found three types of deposits. 1,
Small spherulites (3 to 5 p) that stained pink with filipin
were depicted in both the media and in the intima as
early as age 16. In more advanced ages, the density
of these spherulites increased and they tended to
aggregate extracellularly throughout the intima in
groups of two to five; 2), Elongated structures (tens of
microns) that are stained brilliant white with filipin can
be seen in the middle zone of the intima. The density
of these structures was directly related to the severity
of aortic atherosclerosis and they were detected only
in middle-aged patients. It appears, however, that
these deposits differ from those found in the core of

atherosclerotic plaques (see below), as the former
compared with the latter retained their overall size (10
to 30 p), uniformity (oval elongated), and localization
(middle zone of the intima). 3), Large (100 ,u), irregular
deposits were found mainly in the core of the athero-
sclerotic plaque. The experimental approach used in
this study was novel in two aspects: 1), for avoiding
the use of organic solvents in sample preparation and
2), for viewing the filipin-cholesterol fluorescence on
a single photomicrograph with the use of a fluores-
cence microscope with the triple band pass filter, al-
lowing a better detection and resolution of the filipin-
positive particles.
Unesterified cholesterol particles can be detected
in human aorta as early as age 16 in patients with
histologically normal intima. As shown in Figure 1,
small unesterified cholesterol deposits are widely
scattered in the media. Of interest is our finding that
the earliest cholesterol deposits have been the
spherulites found in the media. Because in older pa-
tients, and in patients with advanced atherosclerosis,
the filipin-positive spherulites in the media remained
sparse, this indicates that these deposits may be tran-
sient and reversible, or they may be accidental and
associated with the individual pathological condition
of the donor. Also of interest is the finding that these
spherulites can be found in the intact intima of arteries
of humans and of animal models. The fact that such
deposits have not been previously reported is the re-
sult of two experimental factors: 1), the minute cho-
lesterol deposits are subjected to rapid dissolution by
organic solvents traditionally used for sample prepa-
ration, which have been avoided in the present study,
and 2), with the previously used optical filters the
minute deposits could not be discerned on the back-
ground of the endogenous tissue fluorescence.
At present, the exact nature of the filipin-positive
spherulites is unknown. However, based on our10 and
other2'3 studies, these small spherical particles can
be speculated to consist of aggregates of the
cholesterol-rich vesicles isolated from human athero-
sclerotic lesions and characterized by Kruth and col-
laborators1 1 and also observed by Simionescu et all
in arterial intima of cholesterol-fed rabbits. In contrast
to membranes of undegraded LDL particles that
have a cholesterol/phospholipid ratio of 0.9:1, in
cholesterol-rich particles the ratio is approximately
2.5:1. The large excess of unesterified cholesterol in
relation to phospholipid does not allow the formation
of 1:1 cholesterol/phospholipid complexes which is
characteristic to most membranes. This may allow the
formation of cholesterol domains that can react with
filipin. The other possibility is that these spherulites
represent unesterified cholesterol crystallites that
Unesterified Cholesterol in Human Aorta 145
AJPJanuiary 1995, Vol. 146, No. 1
were formed through the collapse of cholesterol-rich
particles and the condensation of released choles-
terol molecules.
In patients with early atherosclerosis (eg, Figure 3),
the spherulites containing unesterified cholesterol
tend to accumulate in the lower part of the intima. In
some instances, the circular spherulites form chains
of two to four closely spaced particles. Such a phe-
nomenon suggests that these particles are contained
in the extracellular space. In addition, this may rep-
resent vesicle aggregation, which precedes their col-
lapse and cholesterol release to form crystallites. On
the other hand, small crystallites are also known to
aggregate in an aqueous medium12,13
We have observed an age-related tendency of ac-
cumulation of the spherulites in the intima, particularly
in the lower zone, and the effect was more noticeable
in patients with atherosclerosis. Currently there is no
explanation to this finding, although it may be asso-
ciated with an increased influx and/or decreased ef-
flux of LDL-cholesterol in the arterial walls as age ad-
vances. Another factor to consider is the relative
larger accumulation of the spherulites in the lower
zone of the intima, compared with more superficial
parts. One possible explanation is that the steady
state level of accumulation is equal in all the layers of
the intima but that endothelial and subendothelial lay-
ers are continuously being shed and replaced, thus
creating the impression of less accumulation of
spherulites in the more superficial intimal layers.
The second type of particle containing unesterified
cholesterol was of filipin-positive agglomerates that
are seen mainly in the middle zone of the intima.
These particles appear as a trail of elongated par-
ticles that stain brilliant white with filipin and are lo-
cated in the middle zone of the intima in individuals
already in their twenties (eg, Figure 3A). The aortic
segment of the 28-year-old male demonstrates, in
Figure 3A, intimal thickening with some disruption; the
spherulites are concentrated in the lower and upper
zones of the intima, whereas a trail of elongated
strongly fluorescent white particles is located in the
middle zone. In this figure, the chains of several small
spherulites are possibly in a preaggregation state in
the midzone of the intima, ie, in transition to larger
circular bodies and to the elongated particles. The
larger particles are evidently crystalline, possibly ag-
glomerates of unesterified cholesterol crystals with
calcium phosphate.
Although we have not seen an intermediate state
between Figures 2 and 3A with regard to the transition
from the small spherical particles seen as circular
dots to larger, either elongated or irregularly shaped,
cholesterol crystals and agglomerates (probably be-
146 Sarig et al
AJPJanuary 1995, Vol. 146, No. 1
cause of the limited number of specimens), it is sug-
gested that such state(s) can be found either in a
more extensive survey or in a systematic study of an
animal model.
Of interest is the fact that in all cases the medium
sized, unesterified cholesterol particles were local-
ized in the midzone of the intima (Figures 2 to 4). This
suggests that accumulation of unesterified choles-
terol can occur anywhere within the wall of the artery
but that the irreversible co-crystallization of unesteri-
fied cholesterol with calcium phosphate occurs in a
specific area in the intima. At present we have no
explanation for the susceptibility of the midzone of the
intima to the irreversible crystallization of unesterified
cholesterol; this spatial composition of the lesions,
with large unesterified cholesterol deposits in the
middle zone of the intima may result from the limited
accessibility to this region of agents that govern the
efflux from the arterial wall and the reversibility of un-
esterified cholesterol deposition. Other possible ex-
planations are that this area is the least perfused re-
gion of the intima or that it is the transition zone of the
intact (stationary) intima and the more superficial in-
timal layers that are constantly being replaced.
A third, more advanced form of unesterified cho-
lesterol accumulation was seen in aortas of patients
with advanced atherosclerosis, eg, Figures 4A, B. In
these figures, atherosclerotic plaques have de-
stroyed the lower part of the intima and, along with the
bulk of the plaque, several rather large solid particles
are embedded in the upper part of the core of the
lesion. They can be resolved to small fluorescent par-
ticles and could therefore have been formed by ag-
gregation of small spherical particles of 5 p or less in
diameter. This hypothesis is supported by the simul-
.taneous presence of particles of all sizes at the same
location, ie, the small spherical particles that were
seen as pink dots in Figures 1 to 4 as well as medium
to large sized particles. Aggregates composed of
small cholesterol crystallites are known to have strong
filipin response, whereas large perfect crystals
formed by the mechanism of single crystal growth do
not emit filipin complex fluorescence.6 Seen with the
applied filipin are either aggregates of small choles-
terol crystallites or agglomerates of these crystallites
with different elements of the lesion. This would mean
that if the small dots are aggregates of cholesterol-
rich vesicles, formed by degradation of LDL particles,
then the new order aggregation into large particles
took place before the transition from the solubilized
state of cholesterol to the solid crystalline phase.
The atherosclerotic lesion seen in Figure 4B in the
aorta of a 83-year-old man contains all the elements
seen in the former figures in such density that they are
superimposed on each other. However, the most
prominent characteristic of this structure is the trail of
large, elongated, brilliant white fluorescent particles
in the middle zone of the intima. Comparison of Fig-
ures 3 and 4 reveals remarkably similar structural fea-
tures, despite the differences in the ages of the pa-
tients and extent of the atherosclerosis. The deposits
of the small spherical particles are localized at the
lowest part of the intima close to the elastic boundary.
The upper part of the intima is either completely (Fig-
ure 3A) or partly (Figure 4B) detached, and the mid-
zone of the intima contains numerous large brilliant
white fluorescent particles.
The arrangement of the results of this study in an
order of age-related progressive severity of the ath-
erosclerotic lesion allows us to formulate a preliminary
working hypothesis with respect to a possible physi-
cal aspect of unesterified cholesterol deposition in
arterial wall. Our hypothesis visualizes that choles-
terol deposition is accomplished in two stages of
aggregation. First, after degradation of infiltrating
LDL particles into the arterial wall, unesterified
cholesterol-rich vesicles7 10 are formed. These par-
ticles are too small (approximately 100 to 200 nm) to
be perceived with filipin staining by a light optical mi-
croscope. Some of these particles coalesce to form
the 3- to 5-p spherical particles described as spheru-
lites. At present it is unknown if these particles are
aggregates of vesicles or already small crystallites.
The second event of aggregation occurs when the
spherical basic particles form chains of spherulites,
seen in Figures 2 to 4, mainly in the lower part of the
intima. This stage is still reversible. A more advanced
stage will be formation of thin elongated particles,
followed by more massive agglomeration of irregu-
larly shaped particles containing unesterified choles-
terol and calcium phosphate. In the second stage of
aggregation the cholesterol has already made the
transition to the solid state (ie, post crystallization).
The second stage is irreversible. The processes that
promote reversibility of the formed deposits may be
limited topographically, ie, with respect to the pen-
etration into the intima not being effective on deposits
in the middle zone of the intima.
References
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