Atherosclerosis, 2 1 ( 1975) 179-193 SC Elsevier Scientific Publishing Company,
VARIATIONS
IN
GEOGRAPHIC
HUMAN
179
Amsterdam
AORTIC
- Printed in The Netherlands
FATTY
STREAKS
AMONG
LOCATIONS
CARLOS
RESTREPO*
Department
ofPathology,
RICHARD
AND
Louisiana
E. TRACY
State University
Medical
Center,
New Orleans, La. 701 I2( U.S.A.)
(Received July 3rd, 1974) (Accepted September 25th, 1974)
SUMMARY
Histologic
features
of aortic fatty streaks were examined
in male subjects
aged
IO-39 from Guatemala, Jamaica Negro, Durban Bantu, New Orleans Negro, New Orleans white, Lima and Santiago. These groups were selected from the 19 available in the IAP (International est contrasts
between
Atherosclerosis
Project) because they represented
the great-
the extent of fatty streaks in the young and raised lesions in the
elders. Among these seven groups the extent of raised lesions in the older subjects was significantly but weakly correlated with the type of fatty streaks in the young as measured
by lesion thickness,
content
spindle cells. The severity of leukocytic in fatty streaks
however,
correlated
of demonstrable infiltration
strongly
lipid,
numbers
and prevalence
of foam and
of foci of necrosis
with raised lesions (rank r = 0.90). We
interpret this to imply that the presence of leukocytes and of foci of necrosis in the fatty streak marks its propensity to progress into raised lesions. If this formulation is correct, then the presence of both features in the histology of fatty streaks could be used as a marker in future studies of factors bearing upon the emergence of raised lesions out of fatty streaks.
Key words : Arteriosclerosis
- Atherosclerosis
~ Fattll
streak
- Geographic
patl?ologJ
INTRODUCTION
The relationship of fatty streaks to fibrous plaques is a recurring problem in the pathogenesis of atherosclerosis. There are conflicting data bearing on this question. This investigation was supported by research grants HL-08974 and HL-14496, National Heart and Lung Institute, U.S. Public Health Service. * Visiting Research Professor, permanent address: Departamento de Patologia, Facultad de Medicina, Universidad de Antioquia, Medellin, Colombia. S.A.
180
C. RESTREPO,
Some evidence
supports
the concept
that fibrous
plaques
R. E. TRACY
arise by the progression
lipid accumulation, necrosis and fibromuscular proliferation in fatty streaks. evidence indicates that fibrous plaques arise by an independent process’. In the International geographic advanced
pathology
Project
of atherosclerosis,
atherosclerotic
approximately
Atherosclerosis
(IAP),
a cooperative
study on the
the gross extent of fatty streaks and of more
lesions was assessed in the aortas and coronary
23,000 consecutive
of
Other
autopsy
cases performed
arteries from
in 14 different
geographic
locations2. All the location-race subgroups included in the IAP could be ranked consistently according to the mean extent of raised atherosclerotic lesions (fibrous plaques plus other more advanced lesions) in the intima of the aorta and coronary arteries. When the population groups were ranked in order of atherosclerotic involvement, New Orleans
whites
and Oslo ranked
highest
and Guatemala
and
Durban
Bantu
ranked lowest, and other population groups were intermediates. There was a close relationship between the extent of fatty streaks in the coronary arteries of young persons and the extent of raised lesions in the coronaries at later ages within each location-race
subgroup
studied.
Aortic fatty streaks,
however,
appeared
in early life, and
their gross extent was not closely related to the extent of raised atherosclerotic observed
at later ages4. In general,
young persons
from Negro populations
lesions
had exten-
sive aortic fatty streaking, but older persons from Negro populations had relatively mild involvement by raised lesions in comparison with non-Negro subgroups. These findings imply either that various populations have different propensities to convert aortic fatty streaks into raised lesions or else that some aortic raised lesions may arise independently
of fatty streaks.
This study was undertaken to determine variations in the histologic features of aortic fatty streaks between populations showing different degrees of involvement with raised atherosclerotic lesions. Accordingly, fatty streaks from the thoracic and abdominal aorta of subjects aged IO-39 years from seven selected populations included in the IAP were examined microscopically. Cases included in the present study were the same as used in a similar study of coronary
fatty streakss. A wide range of variabil-
ity was observed regarding microscopic characteristics of aortic lesions grossly classified as fatty streaks. The findings offer some insight into the mechanism of conversion of fatty streaks into fibrous plaques. MATERIALS
AND METHODS
The IAP has made available in a central laboratory 23,207 Sudan IV stained sets of aortas and coronary arteries from 19 location-race groups around the worlds. Specimens in that collection were used for this study. Male cases in age groups 10-19, 20-29, 30-39 whose death was due mainly to trauma, from New Orleans white, New Orleans Negro, Jamaica, Lima, Santiago, Guatemala and Durban Bantu were subjected to random sampling so as to yield 15 cases in each subgroup. These seven populations were chosen because in the IAP they exhibited a wide range of extent of advanced lesions in later decades. Because of deterioration or loss of specimens at
VARIATIONS
IN HUMAN
AORTIC
some stage of processing, subgroup
varied from
FATTY
STREAKS
20 cases were discarded,
fatty streak surrounded
abdominal
segment
embedded
in paraffin
half of the lesion
GEOGRAPHIC
LOCATIONS
so that the number
181
of cases in each
12 to I5 cases.
One block of tissue for microscopic stained
AMONG
study was taken
by uninvolved
of the aorta. and sections
was embedded
Blocks
intima
were then divided
were stained in gelatin,
routinely
sectioned
from the most distinctly
in the thoracic
and also in the
in half.
One
half was
with H & E. The remaining in a cryostat
and stained
for
lipids with Oil Red 0. Grading
procedure
Slides identified only with an accession number were graded sequentially for each aortic segment by two independent judges (the authors). Thus, the judges had no knowledge of age or source of the case being graded. Each judge graded I6 different histologic features using well defined criteria in a previously agreed-upon standard operating procedure. To be objective in the grading of some of the microscopic characteristics
(intimal
5 microphotographs assigned
lipid, leukocytic arranged
infiltrate
by increasing
then from 0 to 5 by comparing
and foam cells) we used a panel of
severity
for each feature.
the most severely involved
Scores were
point of the lesion
with the standard set of photographs so as to locate the interval between appropriate stages of involvement. In some cases the lesion was so small that it was not carried onto the slide during regarding
intimal
processing
of the tissue;
lipid were encountered.
in this way, some cases with grade 0
The final grade assigned
to each lesion was
taken as the mean of the two observers. Each judge also graded
each set of sections
for the following
characteristics:
Oil Red stain. Intimal thickness at the thickest point compared with normal intima (0, I, 2, etc. times thicker than normal); type of lipid (none, fine droplets, coarse or mixed);
predominant
pattern
of lipid (intracellular,
extracellular
or mixed);
medial
lipid (none,
slight, moderate or severe). Smooth muscle cell (spindle cells) proliferation in the intima (none, slight, moderate, severe) and presence of cholesterol clefts, intimal vascularizaH
tion, broad
&
E stain.
necrosis,
hemorrhage,
area of acellularity
Agreement
calcification and smudginess
and fibrous
cap. Necrosis
was defined
as a
seen on H & E.
of gradings
A standard set of 30 slides selected from the cases to be judged with a table of random digits was graded independently by the two judges on two different occasions. Analyses of the results revealed the proportion of inter- and intra-observer agreement. For scale measurements (intimal lipid, leukocytes, intimal thickness and foam cells) equal scores were assigned by the same judge in 59%) and 62% of the two gradings, and they were within one score unit in 987,) and 94% respectively. These results indicated that judges could follow reasonably well the criteria established for the grading procedure. Complete agreement between judges was attained in 51 y/;; of’ the trials on the first grading of the standard set and on 53 x, of the second, and scores
C. RESTREPO,
182 TABLE MEAN
R. E. TRACY
1 EXTENT
SELECTED
OF RAISED
LOCATION-RACE
ATHEROSCLEROTIC GROUPS
AND
LESIONS
BY SEVERAL
IN THE
AORTA
COMBINATIONS
IN MALES OF ARTERIES
25-64 AND
YEARS
IAP
OF AGE
BY
UNWEIGHTED
MEAN
(International
Atherosclerosis
Location-race group New Orleans white New Orleans Negro Jamaica Lima Santiago Guatemala Durban Bantu
Project, 1960-65) ~~____
..~
~___~____
~
Thoracic aorta
Abdominal aorta
Aorta, both segments
Unweighted” mean IAP
ReJbrence rank
12.9
29.9
21.4
18.3
I high
10.7 7.5 7.2 7.3 1.2 5.2
20.2 13.8 12.4 12.0 10.7 9.3
15.4 10.6 9.8 9.6 8.9 1.2
14.5 9.5 8.5 8.2 6.5 6.2
2 high 3 intermediate 4 intermediate 5 low 6 low 7 low
a This measure is derived from the unweighted mean of 40 mean values for raised atherosclerotic lesions for both sexes, 4 age groups and 5 arterial segments, thoracic and abdominal aorta, right, left, and anterior descending coronary arteries in each location-race group (see ref.3).
fell within one score unit in 88 % and 98 “/, respectively.
The level of agreement
between
judges obtained in the grading of the standard set was similar to that reached during the actual grading of all fatty streaks. For analysis, the grade assigned for each lesion was the average of the scores assigned independently by each of the two judges. Intra-observer agreement for characteristics coded as either absent or present was 98 % and 92 % for each judge and inter-observer the two gradings
of the standard
agreement
set. In case of disagreement
was 93 % and 92 % of
the case was accepted
as
positive. Reference arterial
rank
Table I shows the mean extent of raised atherosclerotic lesions for different combinations in males 25-64 years of age and the unweighted mean extent of
involvement
which served as the basis for determining
the reference
rank in the IAP, a
frame of reference designed to compare the location-race groups studied”. All tables and figures in this report are presented with the 7 location-race groups ordered according to their reference rank. Statistical methods The statistical laboratory prepared 7 x 2 and 7 x 3 tables (i.e. Tables 2 and 3) for testing the differences in average scores assigned to the histologic characteristics graded between the 7 location-race groups for each of the 3 age groups (10-19, 20-29 and 30-39 years) and for each of the 2 arterial segments (thoracic aorta and abdominal aorta). The associations between reference rank and some other microscopic items
VARIATIONS
TABLE
IN HUMAN
AORTIC
FATTY
STREAKS
GEOGRAPHIC
183
LOCATIONS
2
NUMBER OF CASES AND MEAN SCORES OF HISTOLOGIC OF MALES
BY LOCATION-RACE
(International
to-19
AMONG
GROUP
Atherosclerosis
New Orleans white
FEATURES
OF FATTY
STREAKS
IN THE THORACIC
AORTA
AND AGE
Project,
1960-65)’
New Orleans Negro Jamaica Lima Santiago Guatemala Durban Bantu
12 I5 I2 I3 I5 I4 I4
2.0 2.2 I.5 I.0 I.1 1.9 1.4
2.1 2.9 2.0 1.3 I .I 2.6 1.9
2.3 2.2 1.9 I.8 I.5 2.1 1.2
2.5 2.4 I .7 0.9 1.2 2.4 1.6
0.9 0.X I .o 0.8 0.4 I.1 0.6
20-29
New Orleans white New Orlans Negro Jamaica Lima Santiago Guatemala Durban Bantu
I4 I5 I4 I5 15 I4 I5
2.5 2.2 2.0 I .I I .? 2.0 2.0
3.2 3.2 2.6 2.5 2.4 2.6 2.1
2.8 3.2 2.5 3.0 2.4 2.5 2.0
3.5 2.8 2.4 I .4 I.7 2.1 2.2
1.7 1.7 1.4 I.4 I.1 1.0 I .o
30-39
New Orleans white New Orleans Negro Jamaica Lima Santiago Guatemala Durban Bantu
15 I5 I5 I3 13 I2 I5
2.6 2.4 1.X 1.7 I .5 2.1 2.2
3.4 3.5 2.1 2.8 2.1 3.1 2.9
3.0 3.3 2.5 3.0 2.1 2.5 I.9
3.2 3.1 2.1 I .7 1.3 2.5 2.6
I.8 I.8 I.5 I.6 I.2 I.6 2.1
:L Analyses of variance (Anovas) revealed significant differences among location-race and age groups (P . 0.01) in each set of values for a given feature. No significant interaction was observed (see ref. r). t1 Scores ranged from 0 to 5. I’ Scores ranged from 0 to 3.
were tested in 3 x 3 tables for every age group, Tests were then computed for the statistical significance of the distributions in these tables. Rank correlation and partial correlation
coefficients
were also computed
for some items’.
RESULTS
There was a high degree of individual variability regarding microscopic severity of aortic lesions grossly classified as fatty streaks. Mean scores for those histologic features graded in scale intervals are presented in Tables 2 and 3. Differences among location-race groups were clearly seen in the thoracic aorta and the abdominal aorta for intimal lipid, intimal thickness, leukocytic infiltration and foam cells (P < 0.01). In general, those location-race groups occupying high places in the reference rank had
184
C. RESTREPO, R. E. TRACY
TABLE 3 MEAN SCORES TION-RACE
OF HISTOLOGIC
GROUP
(International
FEATURES
OF FATTY
STREAKS
IN THE ABDOMINAL
AORTA
OF MALES BY LOCA-
AND AGE
Atherosclerosis
Project, 1960-65)8
Age group
Locution-race group
lntimal thickttessl’
Itttimal lipid”
Leukocytic ittfi/tratet
Foam cells”
Spindle cells’
10-19
New Orleans white New Orleans Negro Jamaica Lima Santiago Guatemala Durban Bantu
2.0 2.1 I.8 I.1 1.3 2.1 I.1
2.8 2.8 2.1 1.3 I .9 2.6 1.5
2.0 2.2 2.0 I .4 2.4 1.6 I.1
2.4 2.2 I .9 1.0 1.3 2.5 1.2
1.1 0.9 1.2 0.8 0.9 1.1 0.9
20-29
New Orleans white New Orleans Negro Jamaica Lima Santiago Guatemala Durban Bantu
2.5 3.1 2.1 1.3 2.1 1.7 1.7
3.1 3.4 2.4 1.4 2.4 2.2 2.3
2.5 2.4 2.5 1.2 2.1 1.6 1.2
2.9 2.5 1.6 0.1 1.7 2.1 I.6
1.9 1.6 1.5 I.1 1.3 1.1 I.1
30-39
New Orleans white New Orleans Negro Jamaica Lima Santiago Guatemala Durban Bank
2.9 3.0 2.3 I.8 2.1 2.4 2.2
3.5 3.4 2.7 2.6 1.9 3.0 2.9
2.6 2.3 2.6 2.1 2.5 2.5 2.0
3.0 3.3 1.9
1.7 1.8 1.6 1.8 I.8 I.9 2.0
I .o I .6 2.5 2.8
a Numbers of cases are equal to those in Table 2. Analyses of variance (Anovas) revealed also significant differences among location-race and age groups (P < 0.01) except in the distribution of scores for spindle cells. No significant interaction was observed (see ref. 7). b Scores ranged from 0 to 5. c Scores ranged from 0 to 3.
higher mean scores than those ranking lower. Lesions from Guatemala and Durban, however, showed higher scores than expected for intimal lipid and foam cells. Leukocytic infiltration was also unexpectedly high in fatty streaks of the thoracic aorta from Lima. Smooth muscle cell proliferation in the intima (called spindle cells in the tables) differed significantly among location-race groups in the thoracic aorta but not in the abdominal aorta. This was the only consistent difference between aorta segments seen in this study. A clear age effect is also observed; the values for histologic features of lesions increased significantly with age for all variables. The thickness of fatty streaks bore a relationship to age almost identical to that for foam cells (Fig. I). The histologic features referred to as leukocytic infiltration are illustrated in Figs. 2, 3, and 4. The leukocytes usually appeared to be lymphocytes or monocytes
VARIATIONS
IN HUMAN
AORTIC
FATTY
STREAKS
GEOGRAPHIC
185
LOCATIONS
A
Age
I
AMONG
14
1.8
2.2
2.6
30
34
Amount intimal lipid (mean index) Fig. I. Age, location-race, aortic segment subgroup means (3 x 7 j 2 -- 42) from Tables 2 and 3. Abdominal and thoracic segments and location-race groups are not given distinctive symbols here because they did not differ significantly in the relationship between these two variables. The correlation coefficient for this plot is 0.90. The dashed regression line represents the “common” slope of parallel lines in the analysis of covariance.
TABLE
4
PARTIAL AORTIC ENCE
CORRELATION FATTY
RANK
STREAKS
GROUPS
(International
COEFFICIENTS AFTER AND
BY
Atherosclerosis
BETWEFN
REGRESSION ARTERIAL
Project,
INTIMAL
ADJUSTMENT
LIPID
FOR AGE;
SCORES
AND
OTHER
MALES
IO-39
YEARS
MEASUREMENTS OF AGE
BY
SEGMENT
1960-1965)
Intimal lipid vs. thicknrss
Iniimal lipid vs. lrukocytic infiltratiorl
Itltimal lipid vs. fowl cdls
Intimal lipid vs. spindle cells
Thorucic aorta high intermediate low
0.41* 0.73* 0..58*
0.28 * 0..52* 0.24*
0.30* 0.68* 0.50*
0.1 I 0.56* 0.36*
Abdominal aorta high intermediate low
0.35* 0.64* 0.65*
0.001 0.52* 0.29*
0.04 0.47* 0.54*
0.40* 0.41* 0.44*
R~~ferencr rtrnk glW!p
*
P
0.01
IN
REFtR-
186
C. RESTREPO, R. E. TRACY
Fig. 2. Fatty streak with a typical leukocytic infiltrate of grade 5 (right) showing the characteristic focal nature of the process and its association with small foci of necrosis. H & E stain; x 100 (enlarged 2. I ,d ).
Fig. 3. Aortic fatty streak showing grade 5 (maximal) severity on the “leukocytic infiltration” scale. Most cells appear to be lymphocytes or monocytes; some foam cells are also present. H & E stain; x 450 (enlarged 2.1 x).
VARIATIONS
Fig. 4. Aortic appear
AORTIC
fatty streak
FATTY
illustrating
to be polymorphonuclear
TABLE RANK
IN HUMAN
STREAKS
grade
leukocytes
AMONG
3 (average)
GEOGRAPHIC
degree of leukocytic
(P). H & E stain:
187
LOCATIONS
infiltration.
450 (enlarged
2. I
Some cells
Y,).
5 CORRELATION
OBTAINED
COEFFICIENTS
BY COMBINING
Necrosis
MEANS
BETWEEN OF
REFERENCE
3 AGE GROUPS
AND
RANK
AND
2 ARTERIAL
RANKS
OF 7 LOCATION-RACE
GROUPS
SEGMENTS
0.90*
Leukocytic infiltrate Spindle
cells
0.90*
0.71*
0.76 *
0.71*
0.63
0.54
0.38
0.69
0.47
0.37
0.63
0.70*
0.99*
-
0.43
0.29
0.51
0.71*
0.93*
0.96*
Intimal thickness lntimal Foam
lipid cells
* Significant
at P
0.73*
0.05
(Fig. 3) although polymorphonuclear leukocytes were also observed (Fig. 4). Foam cells are considered to represent monocytes containing considerable amounts of intracellular lipid (Fig. 3) but were not included in the classification of leukocytic inhltration. Mean scores were combined to obtain for each variable a mean of 6 means (3 age groups, and 2 arterial segments for each location-race group). Rankings of the 7
OF CASES
NUMBERS
aorta
New Orleans white New Orleans Negro Jamaica Lima Santiago Guatemala Durban Bantu
Abdominal
Thoracic aorta New Orleans white New Orleans Negro Jamaica Lima Santiago Guatemala Durban Bantu
41 45 41 41 43 40 44
41 45 41 41 43 40 44
Total No.
group
Lorotion-race
AND
Atherosclerosis
GROUP
ACCORDING
(International
BY LOC’ATION-RACE
6
TABLE
1 1 4 7 IO 2 4 ___
1 1 4 2 12 1 3
none
13 17 18 23 16 11 19
10 16 31 14 15 19
8
fine
1960-65)
SEGMENT
Intin7al
Project, -
ARTERIAL
TO CHARACTERISTICS
OF LIPID
27 21 19 11 17 27 21
32 34 21 8 17 24 22
coarse
27 24 18 8 14 25 23
29 32 21 6 15 26 20
ACCUMULATION
2 2 9 6 2 1 0
2 0 9 10 0 2 4
IN INTIMA
AND
11 15 9 16 14 11 14
7 11 5 19 11 11 15
FATTY
3 2 0 0 1 5 3
7 0 2 2 1 3
1
clqfts
cholesterol
~~
OF AORTIC
extracellular
MEDIA
OF MALES
7 5 13 24 15 11 16
5 4 13 14 16 10 8
t10ne
Medial
STREAKS
21 22 20 6 15 21 17
21 26 17 13 15 22 24
slight
10-39
OF AGE
13 18 8 11 13 8 11
15 15 11 14 12 8 12
abundant
YEARS
z
VARIATIONS
IN HUMAN
AORTIC
FATTY
STREAKS
AMONG
GEOGRAPHIC
LOCATIONS
189
Fig. 5. Aortic fatty streak showing a coarse pattern of lipid deposited inside foam cells. Fine lipid is seen also although it is rather scarce and mainly extracellular. Oil Red 0 stain: I’ 200 (enlarged 2.1 I ).
Fi g. 6. Aortic fatty streak showing a fine pattern of mainly extracellular lipid. Some spindle cells are seen containing lipid. A fibromuscular cap is covering the lesion. Oil Red 0 stain; x 100 (enlarged 2.1 X).
190
C. RESTREPO, R. E. TRACY
TABLE I NUMBERS FATTY
OF CASES
STREAKS
(International
ACCORDING
IN MALES
Atherosclerosis
Location-race
TO THE PRESENCE
10-39
No.
YEARS
New Orleans Negro Jamaica Lima Santiago Guatemala Durban Bantu
HISTOLOGIC
CHARACTERISTICS
IN AORTIC
GROUPS
Project, 1960-65) Necrosis
group Thoracic aorta New Orleans white
OF SEVERAL
OF AGE BY LOCATION-RACE
Fibrous cap
Mural thrombus
41 45 41 41 43 40 44
20 19 15 19 13 11 13
6 15 6 10 4 3 7
2 0 0 1 1 1 0
41 45 41 41 43 40 44
17 13 11 9 12 9 12
13 9 8 4 5 7 9
1 1 2 3 3 0 0
Calcifcation
2 6 4
15 5 4 2
Vascularizalion
Hemorrhage
0
0
1 0 0 0 0 1
0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
Abdominal aorta New Orleans white New Orleans Negro Jamaica Lima Santiago Guatemala Durban Bantu
location-race groups based on the combined measurements were matched with the reference rank based on raised atherosclerotic lesions (Table 4). Spearman rank correlation coefficients computed from these data are shown in Table 57. The severity of leukocytic infiltration (r = 0.90) and of necrosis (r = 0.90) were the variables which could rank these 7 locations most closely following the order of the reference rank indicating
that in this set of measurements
they are the best predictors
of the
later emergence of fibrous plaques. Variations were also noted in the distribution of aortic fatty streaks among these 7 populations according to the morphologic characteristics of the lipid deposited in the intima
and medial layers of the aorta (Table 6). Approximately
one fourth
of the
lesions sampled from Santiago males showed no lipid on microscopic examination, indicating that in this particular population fatty streaks were often so small that they were not properly represented in the Oil Red 0 stained sections. Locations with high degree of involvement by raised atherosclerotic lesions had fatty streaks mainly characterized by coarse droplets of lipid within foam cells and lipid which extended into the medial layer (Fig. 5). On the other hand the populations ranked intermediate and low on raised lesions had more lesions with fine droplets of lipid deposited extracellularly which did not extend into the media (Fig. 6). Cholesterol clefts were seldom seen in any location; however, they were relatively more frequent in the thoracic aorta of New Orleans Negroes and in the abdominal aorta of Guatemalans. However, there
VARIATIONS IN HUMAN AORTIC FATTY STREAKS AMONG GEOGRAPHIC LOCATIONS
was no correlation
between
reference
rank and ranks of location-race
191
groups based on
relative frequency of any of the histologic characteristics in Table 6. Aortic fatty streaks also showed striking differences among populations certain
other microscopic
foci of necrosis.
of this finding ranks the location-race
whites. Nevertheless,
ranking
of fibrous cap in fatty streaks did not correlate
ence rank. Finely stippled
groups in
as the reference rank. Presence of a fibrous cap in fatty in the thoracic aorta in New Orleans Negroes and in the
aorta in New Orleans
the prevalence
(Table 7). Many of the lesions showed microscopic
The relative frequency
a nearly identical fashion streaks was most frequent abdominal
features
for
calcium
deposits
were sometimes
of the populations strongly
found;
by
with the refer-
this event was un-
expectedly frequent in cases from Lima. Its frequency did not correlate significantly with the reference rank. Mural thrombi were rarely seen in any of the populations. Vascularization
and hemorrhage,
very infrequent
in all subgroups.
although
specifically
sought
microscopically,
were
DISCUSSION
Cases included in the present study were chosen from those in the IAP file by a random procedure so as to represent males of selected populations dying of causes not related to atherosclerosis
(“basal”
cases). The relationship
of these cases to the living
populations has been discussed thoroughly elsewheres. The conclusion reached by this study is that the rankings race groups by mean extent of raised atherosclerotic with several histological characteristics of aortic Notable
among these are thickness
of lesion, amount
of the seven location-
lesions in older ages is associated fatty streaks in young subjects. of lipid, numbers
of leukocytes,
spindle cells and foam cells, and the presence of foci of necrosis (Table 5). As shown by the correlation coefficients of Table 5, these variables fall naturally into two clusters. Thickness,
lipid, and foam cells are strongly
intercorrelated,
while spindle cells, leuko-
cytes, necrosis, and reference rank form another cluster, and the two clusters are poorly correlated with each other. A previous study of these same 295 subjects5 reported findings on histologic features
of fatty streaks
in the coronary
arteries.
The features
of coronary
arteries
which tended to rank the location-race groups in the same order as raised lesions were thickness of intimal lesion, cellularity (leukocytic), and amount of lipid. There is no disagreement between those findings and findings on aortic fatty streaks reported herein. In all these ways of measuring the average histologic severity of early atherosclerotic lesions, emergence of raised lesions in later life is to a great extent foreshadowed by youthful fatty streaks. As pointed out by McGill 1$4there are certain discrepancies in this association which deserve emphasis. Generally speaking, young Negro males from several populations developed considerably more extensive aortic fatty streaks than non-Negro males. In this microscopic study we found also that Negroes had excessive lipid content in aortic fatty streaks. Furthermore, in this particular set of
192
C. RESTREPO,
cases it was also found that Guatemalans
had excessive amounts
R. E. TRACY
of lipid in aortic fatty
streaks in relation to the low tendency to develop raised lesions at older ages. These discrepancies suggest that some populations (i.e., New Orleans Negro, Durban Bantu and Guatemala)
have fatty streaks which do not progress or progress
raised lesions as compared to other populations. The leukocytic content of fatty streaks was found Geer et al.j, to show a close association Nevertheless,
as pointed
out by Geer
in this study, and in that of
with the later emergence et al., and
more slowly into
confirmed
of raised lesions.
in the present
study,
“Cellular infiltration occurs only when intimal lipid is moderate or abundant, but intimal lipid may be abundant without cellular infiltration.” The prevalence of necrosis in aortic fatty streaks extent of raised lesions.
is another
feature
showing
Thus, it appears that it is not solely the quantity that determines
its progression
since intimal
close association
with the
of intimal lipid in the fatty streak
lipid can be both extensive
(in terms of
gross extent of intimal surface involved with fatty streaks) and high in density per unit of arterial wall (as judged histologically) without progressing rapidly to fibrous plaques. Some other quality of the fatty streak (perhaps associated with cellular infiltration and foci of necrosis
as found
in this study) is responsible
for the generation
raised lesions. More precise identification or definition of this quality important missing link in the pathogenesis of atherosclerosis. The unidentified a predisposition ting monocytes
quality
to progress
associated
with leukocytic
would
cellular infiltration
of
fill an
and with
to raised lesions could reside in the lipid, in the infiltra-
and macrophages
that become foam cells, or in the mesenchymal
of the arterial wall. It is possible that under some environmental
conditions,
tissue
the lipid is
more injurious or more difficult to be removed by the monocytes. The monocytes and foam cells may, under some conditions, be less efficient in the ingestion and removal of lipid. Smooth muscle and connective tissue may, also under some conditions, react with a stronger proliferative response than under other conditions. There might also be humoral differences among these groups that would be responsible for the differences in leukocytic response. For example, the well established differences in prevalence of the various risk factors (particularly hyperlipidemia, hypertension, and cigarette smoking) might affect lipid influx, metabolism, and efflux or the efficiency with which the macrophages dispose of phagocytosed lipid. Some other findings also deserve some consideration. The rarity with which mural thrombus was found in aortic fatty streaks and the conspicuous absence of vascularization and hemorrhage tend to add weight against hypotheses of a major role for these processes in the early development of atherosclerotic lesionsgvrO. In addition, calcification does not seem to be related to the propensity to convert fatty streaks into fibrous plaques. The finding of an unexpectedly high frequency of calcification in Lima could be related to the well established fact that water hardness (and calcium content of the drinking water) is extremely high in this locationll. From our findings we conclude that (1) raised lesions in older ages are partially predicted by fatty streaks in the young, (2) some location-race groups do not develop
VARIATIONS
IN HUMAN
AORTIC
the degree of raised lesions young,
and (3) leukocytic
location-race
groups
FATTY
expected
infiltration
STREAKS
AMONG
from the extent
GEOGRAPHIC
of fatty streaking
and foci of necrosis
with the higher propensities
LOCATIONS
193
seen in the
in fatty streaks mark those
to develop
raised lesions.
ACKNOWLEDGEMENTS
We are indebted
to Drs. Henry C. McGill,
Jr., Jack P. Strong
and Douglas
A.
Eggen for constructive criticism and encouragement during the development of the study and preparation of the manuscript. The valuable assistance of Mrs. Martha T. McMurry and Mrs. Vivian T. Toca regarding acknowledged.
the statistical
analyses
is also gratefully
REFERENCES
I MCGILL, JR., H. C., The lesion, Proccvdings of‘ /he Thitd Infernutioml Symposium on Athrro.sc/wosis, West Berlin, October 1973, In press. 2 MCGILL, JR., H. C., ARIAS-STELLA, J., CARBONELL, L. M.. CORREA, P., DEVEYRA, JR., E. A., DONOSO, S., EGGEN, D. A., GALINDO, L., GUZMAN, M. A., LKHTENBERGER, E.. LOKEN, A. C., MCGARRY, P. A., MCMAHAN, C. A., MONTENEGRO, M. R., Moossu, J., PEREZ-TAMAYO, R., RESTREPO, C., ROBERTSON, W. B., SALAS, J., SOLBERG, L. A., STKONC, J. P., TEJADA, C. AND WAINWRIGHT, J., General findings of the international atherosclerosis project, Lab. /nvr.st., 18 (1968) 498. 3 TEJADA, C., STRONG, J. P., MONTENEGRO, M. R., RESTREPO, C. AND SOLBERG, L. A., Distribution of coronary and aortic atherosclerosis by geographic location, race and sex, Lub. Inwsr., 18 ( 1968) 509. 4 MCGILL, JR., H. C., Fatty streaks in the coronary arteries and aorta, Lab. Imvsf., 18 (1968) 560. 5 GEER, J. C., MCGILL, JR., H. C., ROBERTSON, W. B. AND STRONG, J. P., Histologic characteristics of coronary artery fatty streaks, Lob. Invest., 18 (1967) 565. 6 GUZMAN, M. D., MCMAHAN, C. A., MCGILL, JR., H. C., STRohti, J. P., TEJADA, C., RESTREPO, C., EGGEN, D. A., ROBERTSON, W. B. AND SOLBERG, L. A., Selected methodologic aspects of the international atherosclerosis project, Lab. Invrst., 18 (1968) 479. 7 SNEDECOR, G. W. AND COCHRAN, W. G., Statistic’ul Mefhocls, 6th edition, The Iowa State University Press, Ames, Iowa, 1967, pp. 166-170. 8 MCMAHAN, C. A., Autopsied cases by age, sex and “race”, Lab. Invest., 18 (1968) 468. 9 HAUST, M. D., The morphogenesis and fate of potential and early atherosclerotic lesions, Hutmt~ f’urhology. 2 ( I97 I ) I IO HAUST, M. D. AND MORE, R. H., Development of modern theories on the pathogenesis of atherosclerosis. In: R. W. WISSLER AND J. C. GEER (Eds.), Puthogu7esi.r oj’Afhuosclwosi.q The Williams and Wilkins Co., Baltimore, Md., 1972, pp. I-19. 1 I STRONG, J. P., CORREA, P. AND SOLBERG, L. A.. Water hardness and atherosclerosis, Lab. Imwr., I8 ( 1968) 620.
VARIATIONS
IN
GEOGRAPHIC
HUMAN
179
Amsterdam
AORTIC
- Printed in The Netherlands
FATTY
STREAKS
AMONG
LOCATIONS
CARLOS
RESTREPO*
Department
ofPathology,
RICHARD
AND
Louisiana
E. TRACY
State University
Medical
Center,
New Orleans, La. 701 I2( U.S.A.)
(Received July 3rd, 1974) (Accepted September 25th, 1974)
SUMMARY
Histologic
features
of aortic fatty streaks were examined
in male subjects
aged
IO-39 from Guatemala, Jamaica Negro, Durban Bantu, New Orleans Negro, New Orleans white, Lima and Santiago. These groups were selected from the 19 available in the IAP (International est contrasts
between
Atherosclerosis
Project) because they represented
the great-
the extent of fatty streaks in the young and raised lesions in the
elders. Among these seven groups the extent of raised lesions in the older subjects was significantly but weakly correlated with the type of fatty streaks in the young as measured
by lesion thickness,
content
spindle cells. The severity of leukocytic in fatty streaks
however,
correlated
of demonstrable infiltration
strongly
lipid,
numbers
and prevalence
of foam and
of foci of necrosis
with raised lesions (rank r = 0.90). We
interpret this to imply that the presence of leukocytes and of foci of necrosis in the fatty streak marks its propensity to progress into raised lesions. If this formulation is correct, then the presence of both features in the histology of fatty streaks could be used as a marker in future studies of factors bearing upon the emergence of raised lesions out of fatty streaks.
Key words : Arteriosclerosis
- Atherosclerosis
~ Fattll
streak
- Geographic
patl?ologJ
INTRODUCTION
The relationship of fatty streaks to fibrous plaques is a recurring problem in the pathogenesis of atherosclerosis. There are conflicting data bearing on this question. This investigation was supported by research grants HL-08974 and HL-14496, National Heart and Lung Institute, U.S. Public Health Service. * Visiting Research Professor, permanent address: Departamento de Patologia, Facultad de Medicina, Universidad de Antioquia, Medellin, Colombia. S.A.
180
C. RESTREPO,
Some evidence
supports
the concept
that fibrous
plaques
R. E. TRACY
arise by the progression
lipid accumulation, necrosis and fibromuscular proliferation in fatty streaks. evidence indicates that fibrous plaques arise by an independent process’. In the International geographic advanced
pathology
Project
of atherosclerosis,
atherosclerotic
approximately
Atherosclerosis
(IAP),
a cooperative
study on the
the gross extent of fatty streaks and of more
lesions was assessed in the aortas and coronary
23,000 consecutive
of
Other
autopsy
cases performed
arteries from
in 14 different
geographic
locations2. All the location-race subgroups included in the IAP could be ranked consistently according to the mean extent of raised atherosclerotic lesions (fibrous plaques plus other more advanced lesions) in the intima of the aorta and coronary arteries. When the population groups were ranked in order of atherosclerotic involvement, New Orleans
whites
and Oslo ranked
highest
and Guatemala
and
Durban
Bantu
ranked lowest, and other population groups were intermediates. There was a close relationship between the extent of fatty streaks in the coronary arteries of young persons and the extent of raised lesions in the coronaries at later ages within each location-race
subgroup
studied.
Aortic fatty streaks,
however,
appeared
in early life, and
their gross extent was not closely related to the extent of raised atherosclerotic observed
at later ages4. In general,
young persons
from Negro populations
lesions
had exten-
sive aortic fatty streaking, but older persons from Negro populations had relatively mild involvement by raised lesions in comparison with non-Negro subgroups. These findings imply either that various populations have different propensities to convert aortic fatty streaks into raised lesions or else that some aortic raised lesions may arise independently
of fatty streaks.
This study was undertaken to determine variations in the histologic features of aortic fatty streaks between populations showing different degrees of involvement with raised atherosclerotic lesions. Accordingly, fatty streaks from the thoracic and abdominal aorta of subjects aged IO-39 years from seven selected populations included in the IAP were examined microscopically. Cases included in the present study were the same as used in a similar study of coronary
fatty streakss. A wide range of variabil-
ity was observed regarding microscopic characteristics of aortic lesions grossly classified as fatty streaks. The findings offer some insight into the mechanism of conversion of fatty streaks into fibrous plaques. MATERIALS
AND METHODS
The IAP has made available in a central laboratory 23,207 Sudan IV stained sets of aortas and coronary arteries from 19 location-race groups around the worlds. Specimens in that collection were used for this study. Male cases in age groups 10-19, 20-29, 30-39 whose death was due mainly to trauma, from New Orleans white, New Orleans Negro, Jamaica, Lima, Santiago, Guatemala and Durban Bantu were subjected to random sampling so as to yield 15 cases in each subgroup. These seven populations were chosen because in the IAP they exhibited a wide range of extent of advanced lesions in later decades. Because of deterioration or loss of specimens at
VARIATIONS
IN HUMAN
AORTIC
some stage of processing, subgroup
varied from
FATTY
STREAKS
20 cases were discarded,
fatty streak surrounded
abdominal
segment
embedded
in paraffin
half of the lesion
GEOGRAPHIC
LOCATIONS
so that the number
181
of cases in each
12 to I5 cases.
One block of tissue for microscopic stained
AMONG
study was taken
by uninvolved
of the aorta. and sections
was embedded
Blocks
intima
were then divided
were stained in gelatin,
routinely
sectioned
from the most distinctly
in the thoracic
and also in the
in half.
One
half was
with H & E. The remaining in a cryostat
and stained
for
lipids with Oil Red 0. Grading
procedure
Slides identified only with an accession number were graded sequentially for each aortic segment by two independent judges (the authors). Thus, the judges had no knowledge of age or source of the case being graded. Each judge graded I6 different histologic features using well defined criteria in a previously agreed-upon standard operating procedure. To be objective in the grading of some of the microscopic characteristics
(intimal
5 microphotographs assigned
lipid, leukocytic arranged
infiltrate
by increasing
then from 0 to 5 by comparing
and foam cells) we used a panel of
severity
for each feature.
the most severely involved
Scores were
point of the lesion
with the standard set of photographs so as to locate the interval between appropriate stages of involvement. In some cases the lesion was so small that it was not carried onto the slide during regarding
intimal
processing
of the tissue;
lipid were encountered.
in this way, some cases with grade 0
The final grade assigned
to each lesion was
taken as the mean of the two observers. Each judge also graded
each set of sections
for the following
characteristics:
Oil Red stain. Intimal thickness at the thickest point compared with normal intima (0, I, 2, etc. times thicker than normal); type of lipid (none, fine droplets, coarse or mixed);
predominant
pattern
of lipid (intracellular,
extracellular
or mixed);
medial
lipid (none,
slight, moderate or severe). Smooth muscle cell (spindle cells) proliferation in the intima (none, slight, moderate, severe) and presence of cholesterol clefts, intimal vascularizaH
tion, broad
&
E stain.
necrosis,
hemorrhage,
area of acellularity
Agreement
calcification and smudginess
and fibrous
cap. Necrosis
was defined
as a
seen on H & E.
of gradings
A standard set of 30 slides selected from the cases to be judged with a table of random digits was graded independently by the two judges on two different occasions. Analyses of the results revealed the proportion of inter- and intra-observer agreement. For scale measurements (intimal lipid, leukocytes, intimal thickness and foam cells) equal scores were assigned by the same judge in 59%) and 62% of the two gradings, and they were within one score unit in 987,) and 94% respectively. These results indicated that judges could follow reasonably well the criteria established for the grading procedure. Complete agreement between judges was attained in 51 y/;; of’ the trials on the first grading of the standard set and on 53 x, of the second, and scores
C. RESTREPO,
182 TABLE MEAN
R. E. TRACY
1 EXTENT
SELECTED
OF RAISED
LOCATION-RACE
ATHEROSCLEROTIC GROUPS
AND
LESIONS
BY SEVERAL
IN THE
AORTA
COMBINATIONS
IN MALES OF ARTERIES
25-64 AND
YEARS
IAP
OF AGE
BY
UNWEIGHTED
MEAN
(International
Atherosclerosis
Location-race group New Orleans white New Orleans Negro Jamaica Lima Santiago Guatemala Durban Bantu
Project, 1960-65) ~~____
..~
~___~____
~
Thoracic aorta
Abdominal aorta
Aorta, both segments
Unweighted” mean IAP
ReJbrence rank
12.9
29.9
21.4
18.3
I high
10.7 7.5 7.2 7.3 1.2 5.2
20.2 13.8 12.4 12.0 10.7 9.3
15.4 10.6 9.8 9.6 8.9 1.2
14.5 9.5 8.5 8.2 6.5 6.2
2 high 3 intermediate 4 intermediate 5 low 6 low 7 low
a This measure is derived from the unweighted mean of 40 mean values for raised atherosclerotic lesions for both sexes, 4 age groups and 5 arterial segments, thoracic and abdominal aorta, right, left, and anterior descending coronary arteries in each location-race group (see ref.3).
fell within one score unit in 88 % and 98 “/, respectively.
The level of agreement
between
judges obtained in the grading of the standard set was similar to that reached during the actual grading of all fatty streaks. For analysis, the grade assigned for each lesion was the average of the scores assigned independently by each of the two judges. Intra-observer agreement for characteristics coded as either absent or present was 98 % and 92 % for each judge and inter-observer the two gradings
of the standard
agreement
set. In case of disagreement
was 93 % and 92 % of
the case was accepted
as
positive. Reference arterial
rank
Table I shows the mean extent of raised atherosclerotic lesions for different combinations in males 25-64 years of age and the unweighted mean extent of
involvement
which served as the basis for determining
the reference
rank in the IAP, a
frame of reference designed to compare the location-race groups studied”. All tables and figures in this report are presented with the 7 location-race groups ordered according to their reference rank. Statistical methods The statistical laboratory prepared 7 x 2 and 7 x 3 tables (i.e. Tables 2 and 3) for testing the differences in average scores assigned to the histologic characteristics graded between the 7 location-race groups for each of the 3 age groups (10-19, 20-29 and 30-39 years) and for each of the 2 arterial segments (thoracic aorta and abdominal aorta). The associations between reference rank and some other microscopic items
VARIATIONS
TABLE
IN HUMAN
AORTIC
FATTY
STREAKS
GEOGRAPHIC
183
LOCATIONS
2
NUMBER OF CASES AND MEAN SCORES OF HISTOLOGIC OF MALES
BY LOCATION-RACE
(International
to-19
AMONG
GROUP
Atherosclerosis
New Orleans white
FEATURES
OF FATTY
STREAKS
IN THE THORACIC
AORTA
AND AGE
Project,
1960-65)’
New Orleans Negro Jamaica Lima Santiago Guatemala Durban Bantu
12 I5 I2 I3 I5 I4 I4
2.0 2.2 I.5 I.0 I.1 1.9 1.4
2.1 2.9 2.0 1.3 I .I 2.6 1.9
2.3 2.2 1.9 I.8 I.5 2.1 1.2
2.5 2.4 I .7 0.9 1.2 2.4 1.6
0.9 0.X I .o 0.8 0.4 I.1 0.6
20-29
New Orleans white New Orlans Negro Jamaica Lima Santiago Guatemala Durban Bantu
I4 I5 I4 I5 15 I4 I5
2.5 2.2 2.0 I .I I .? 2.0 2.0
3.2 3.2 2.6 2.5 2.4 2.6 2.1
2.8 3.2 2.5 3.0 2.4 2.5 2.0
3.5 2.8 2.4 I .4 I.7 2.1 2.2
1.7 1.7 1.4 I.4 I.1 1.0 I .o
30-39
New Orleans white New Orleans Negro Jamaica Lima Santiago Guatemala Durban Bantu
15 I5 I5 I3 13 I2 I5
2.6 2.4 1.X 1.7 I .5 2.1 2.2
3.4 3.5 2.1 2.8 2.1 3.1 2.9
3.0 3.3 2.5 3.0 2.1 2.5 I.9
3.2 3.1 2.1 I .7 1.3 2.5 2.6
I.8 I.8 I.5 I.6 I.2 I.6 2.1
:L Analyses of variance (Anovas) revealed significant differences among location-race and age groups (P . 0.01) in each set of values for a given feature. No significant interaction was observed (see ref. r). t1 Scores ranged from 0 to 5. I’ Scores ranged from 0 to 3.
were tested in 3 x 3 tables for every age group, Tests were then computed for the statistical significance of the distributions in these tables. Rank correlation and partial correlation
coefficients
were also computed
for some items’.
RESULTS
There was a high degree of individual variability regarding microscopic severity of aortic lesions grossly classified as fatty streaks. Mean scores for those histologic features graded in scale intervals are presented in Tables 2 and 3. Differences among location-race groups were clearly seen in the thoracic aorta and the abdominal aorta for intimal lipid, intimal thickness, leukocytic infiltration and foam cells (P < 0.01). In general, those location-race groups occupying high places in the reference rank had
184
C. RESTREPO, R. E. TRACY
TABLE 3 MEAN SCORES TION-RACE
OF HISTOLOGIC
GROUP
(International
FEATURES
OF FATTY
STREAKS
IN THE ABDOMINAL
AORTA
OF MALES BY LOCA-
AND AGE
Atherosclerosis
Project, 1960-65)8
Age group
Locution-race group
lntimal thickttessl’
Itttimal lipid”
Leukocytic ittfi/tratet
Foam cells”
Spindle cells’
10-19
New Orleans white New Orleans Negro Jamaica Lima Santiago Guatemala Durban Bantu
2.0 2.1 I.8 I.1 1.3 2.1 I.1
2.8 2.8 2.1 1.3 I .9 2.6 1.5
2.0 2.2 2.0 I .4 2.4 1.6 I.1
2.4 2.2 I .9 1.0 1.3 2.5 1.2
1.1 0.9 1.2 0.8 0.9 1.1 0.9
20-29
New Orleans white New Orleans Negro Jamaica Lima Santiago Guatemala Durban Bantu
2.5 3.1 2.1 1.3 2.1 1.7 1.7
3.1 3.4 2.4 1.4 2.4 2.2 2.3
2.5 2.4 2.5 1.2 2.1 1.6 1.2
2.9 2.5 1.6 0.1 1.7 2.1 I.6
1.9 1.6 1.5 I.1 1.3 1.1 I.1
30-39
New Orleans white New Orleans Negro Jamaica Lima Santiago Guatemala Durban Bank
2.9 3.0 2.3 I.8 2.1 2.4 2.2
3.5 3.4 2.7 2.6 1.9 3.0 2.9
2.6 2.3 2.6 2.1 2.5 2.5 2.0
3.0 3.3 1.9
1.7 1.8 1.6 1.8 I.8 I.9 2.0
I .o I .6 2.5 2.8
a Numbers of cases are equal to those in Table 2. Analyses of variance (Anovas) revealed also significant differences among location-race and age groups (P < 0.01) except in the distribution of scores for spindle cells. No significant interaction was observed (see ref. 7). b Scores ranged from 0 to 5. c Scores ranged from 0 to 3.
higher mean scores than those ranking lower. Lesions from Guatemala and Durban, however, showed higher scores than expected for intimal lipid and foam cells. Leukocytic infiltration was also unexpectedly high in fatty streaks of the thoracic aorta from Lima. Smooth muscle cell proliferation in the intima (called spindle cells in the tables) differed significantly among location-race groups in the thoracic aorta but not in the abdominal aorta. This was the only consistent difference between aorta segments seen in this study. A clear age effect is also observed; the values for histologic features of lesions increased significantly with age for all variables. The thickness of fatty streaks bore a relationship to age almost identical to that for foam cells (Fig. I). The histologic features referred to as leukocytic infiltration are illustrated in Figs. 2, 3, and 4. The leukocytes usually appeared to be lymphocytes or monocytes
VARIATIONS
IN HUMAN
AORTIC
FATTY
STREAKS
GEOGRAPHIC
185
LOCATIONS
A
Age
I
AMONG
14
1.8
2.2
2.6
30
34
Amount intimal lipid (mean index) Fig. I. Age, location-race, aortic segment subgroup means (3 x 7 j 2 -- 42) from Tables 2 and 3. Abdominal and thoracic segments and location-race groups are not given distinctive symbols here because they did not differ significantly in the relationship between these two variables. The correlation coefficient for this plot is 0.90. The dashed regression line represents the “common” slope of parallel lines in the analysis of covariance.
TABLE
4
PARTIAL AORTIC ENCE
CORRELATION FATTY
RANK
STREAKS
GROUPS
(International
COEFFICIENTS AFTER AND
BY
Atherosclerosis
BETWEFN
REGRESSION ARTERIAL
Project,
INTIMAL
ADJUSTMENT
LIPID
FOR AGE;
SCORES
AND
OTHER
MALES
IO-39
YEARS
MEASUREMENTS OF AGE
BY
SEGMENT
1960-1965)
Intimal lipid vs. thicknrss
Iniimal lipid vs. lrukocytic infiltratiorl
Itltimal lipid vs. fowl cdls
Intimal lipid vs. spindle cells
Thorucic aorta high intermediate low
0.41* 0.73* 0..58*
0.28 * 0..52* 0.24*
0.30* 0.68* 0.50*
0.1 I 0.56* 0.36*
Abdominal aorta high intermediate low
0.35* 0.64* 0.65*
0.001 0.52* 0.29*
0.04 0.47* 0.54*
0.40* 0.41* 0.44*
R~~ferencr rtrnk glW!p
*
P
0.01
IN
REFtR-
186
C. RESTREPO, R. E. TRACY
Fig. 2. Fatty streak with a typical leukocytic infiltrate of grade 5 (right) showing the characteristic focal nature of the process and its association with small foci of necrosis. H & E stain; x 100 (enlarged 2. I ,d ).
Fig. 3. Aortic fatty streak showing grade 5 (maximal) severity on the “leukocytic infiltration” scale. Most cells appear to be lymphocytes or monocytes; some foam cells are also present. H & E stain; x 450 (enlarged 2.1 x).
VARIATIONS
Fig. 4. Aortic appear
AORTIC
fatty streak
FATTY
illustrating
to be polymorphonuclear
TABLE RANK
IN HUMAN
STREAKS
grade
leukocytes
AMONG
3 (average)
GEOGRAPHIC
degree of leukocytic
(P). H & E stain:
187
LOCATIONS
infiltration.
450 (enlarged
2. I
Some cells
Y,).
5 CORRELATION
OBTAINED
COEFFICIENTS
BY COMBINING
Necrosis
MEANS
BETWEEN OF
REFERENCE
3 AGE GROUPS
AND
RANK
AND
2 ARTERIAL
RANKS
OF 7 LOCATION-RACE
GROUPS
SEGMENTS
0.90*
Leukocytic infiltrate Spindle
cells
0.90*
0.71*
0.76 *
0.71*
0.63
0.54
0.38
0.69
0.47
0.37
0.63
0.70*
0.99*
-
0.43
0.29
0.51
0.71*
0.93*
0.96*
Intimal thickness lntimal Foam
lipid cells
* Significant
at P
0.73*
0.05
(Fig. 3) although polymorphonuclear leukocytes were also observed (Fig. 4). Foam cells are considered to represent monocytes containing considerable amounts of intracellular lipid (Fig. 3) but were not included in the classification of leukocytic inhltration. Mean scores were combined to obtain for each variable a mean of 6 means (3 age groups, and 2 arterial segments for each location-race group). Rankings of the 7
OF CASES
NUMBERS
aorta
New Orleans white New Orleans Negro Jamaica Lima Santiago Guatemala Durban Bantu
Abdominal
Thoracic aorta New Orleans white New Orleans Negro Jamaica Lima Santiago Guatemala Durban Bantu
41 45 41 41 43 40 44
41 45 41 41 43 40 44
Total No.
group
Lorotion-race
AND
Atherosclerosis
GROUP
ACCORDING
(International
BY LOC’ATION-RACE
6
TABLE
1 1 4 7 IO 2 4 ___
1 1 4 2 12 1 3
none
13 17 18 23 16 11 19
10 16 31 14 15 19
8
fine
1960-65)
SEGMENT
Intin7al
Project, -
ARTERIAL
TO CHARACTERISTICS
OF LIPID
27 21 19 11 17 27 21
32 34 21 8 17 24 22
coarse
27 24 18 8 14 25 23
29 32 21 6 15 26 20
ACCUMULATION
2 2 9 6 2 1 0
2 0 9 10 0 2 4
IN INTIMA
AND
11 15 9 16 14 11 14
7 11 5 19 11 11 15
FATTY
3 2 0 0 1 5 3
7 0 2 2 1 3
1
clqfts
cholesterol
~~
OF AORTIC
extracellular
MEDIA
OF MALES
7 5 13 24 15 11 16
5 4 13 14 16 10 8
t10ne
Medial
STREAKS
21 22 20 6 15 21 17
21 26 17 13 15 22 24
slight
10-39
OF AGE
13 18 8 11 13 8 11
15 15 11 14 12 8 12
abundant
YEARS
z
VARIATIONS
IN HUMAN
AORTIC
FATTY
STREAKS
AMONG
GEOGRAPHIC
LOCATIONS
189
Fig. 5. Aortic fatty streak showing a coarse pattern of lipid deposited inside foam cells. Fine lipid is seen also although it is rather scarce and mainly extracellular. Oil Red 0 stain: I’ 200 (enlarged 2.1 I ).
Fi g. 6. Aortic fatty streak showing a fine pattern of mainly extracellular lipid. Some spindle cells are seen containing lipid. A fibromuscular cap is covering the lesion. Oil Red 0 stain; x 100 (enlarged 2.1 X).
190
C. RESTREPO, R. E. TRACY
TABLE I NUMBERS FATTY
OF CASES
STREAKS
(International
ACCORDING
IN MALES
Atherosclerosis
Location-race
TO THE PRESENCE
10-39
No.
YEARS
New Orleans Negro Jamaica Lima Santiago Guatemala Durban Bantu
HISTOLOGIC
CHARACTERISTICS
IN AORTIC
GROUPS
Project, 1960-65) Necrosis
group Thoracic aorta New Orleans white
OF SEVERAL
OF AGE BY LOCATION-RACE
Fibrous cap
Mural thrombus
41 45 41 41 43 40 44
20 19 15 19 13 11 13
6 15 6 10 4 3 7
2 0 0 1 1 1 0
41 45 41 41 43 40 44
17 13 11 9 12 9 12
13 9 8 4 5 7 9
1 1 2 3 3 0 0
Calcifcation
2 6 4
15 5 4 2
Vascularizalion
Hemorrhage
0
0
1 0 0 0 0 1
0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
Abdominal aorta New Orleans white New Orleans Negro Jamaica Lima Santiago Guatemala Durban Bantu
location-race groups based on the combined measurements were matched with the reference rank based on raised atherosclerotic lesions (Table 4). Spearman rank correlation coefficients computed from these data are shown in Table 57. The severity of leukocytic infiltration (r = 0.90) and of necrosis (r = 0.90) were the variables which could rank these 7 locations most closely following the order of the reference rank indicating
that in this set of measurements
they are the best predictors
of the
later emergence of fibrous plaques. Variations were also noted in the distribution of aortic fatty streaks among these 7 populations according to the morphologic characteristics of the lipid deposited in the intima
and medial layers of the aorta (Table 6). Approximately
one fourth
of the
lesions sampled from Santiago males showed no lipid on microscopic examination, indicating that in this particular population fatty streaks were often so small that they were not properly represented in the Oil Red 0 stained sections. Locations with high degree of involvement by raised atherosclerotic lesions had fatty streaks mainly characterized by coarse droplets of lipid within foam cells and lipid which extended into the medial layer (Fig. 5). On the other hand the populations ranked intermediate and low on raised lesions had more lesions with fine droplets of lipid deposited extracellularly which did not extend into the media (Fig. 6). Cholesterol clefts were seldom seen in any location; however, they were relatively more frequent in the thoracic aorta of New Orleans Negroes and in the abdominal aorta of Guatemalans. However, there
VARIATIONS IN HUMAN AORTIC FATTY STREAKS AMONG GEOGRAPHIC LOCATIONS
was no correlation
between
reference
rank and ranks of location-race
191
groups based on
relative frequency of any of the histologic characteristics in Table 6. Aortic fatty streaks also showed striking differences among populations certain
other microscopic
foci of necrosis.
of this finding ranks the location-race
whites. Nevertheless,
ranking
of fibrous cap in fatty streaks did not correlate
ence rank. Finely stippled
groups in
as the reference rank. Presence of a fibrous cap in fatty in the thoracic aorta in New Orleans Negroes and in the
aorta in New Orleans
the prevalence
(Table 7). Many of the lesions showed microscopic
The relative frequency
a nearly identical fashion streaks was most frequent abdominal
features
for
calcium
deposits
were sometimes
of the populations strongly
found;
by
with the refer-
this event was un-
expectedly frequent in cases from Lima. Its frequency did not correlate significantly with the reference rank. Mural thrombi were rarely seen in any of the populations. Vascularization
and hemorrhage,
very infrequent
in all subgroups.
although
specifically
sought
microscopically,
were
DISCUSSION
Cases included in the present study were chosen from those in the IAP file by a random procedure so as to represent males of selected populations dying of causes not related to atherosclerosis
(“basal”
cases). The relationship
of these cases to the living
populations has been discussed thoroughly elsewheres. The conclusion reached by this study is that the rankings race groups by mean extent of raised atherosclerotic with several histological characteristics of aortic Notable
among these are thickness
of lesion, amount
of the seven location-
lesions in older ages is associated fatty streaks in young subjects. of lipid, numbers
of leukocytes,
spindle cells and foam cells, and the presence of foci of necrosis (Table 5). As shown by the correlation coefficients of Table 5, these variables fall naturally into two clusters. Thickness,
lipid, and foam cells are strongly
intercorrelated,
while spindle cells, leuko-
cytes, necrosis, and reference rank form another cluster, and the two clusters are poorly correlated with each other. A previous study of these same 295 subjects5 reported findings on histologic features
of fatty streaks
in the coronary
arteries.
The features
of coronary
arteries
which tended to rank the location-race groups in the same order as raised lesions were thickness of intimal lesion, cellularity (leukocytic), and amount of lipid. There is no disagreement between those findings and findings on aortic fatty streaks reported herein. In all these ways of measuring the average histologic severity of early atherosclerotic lesions, emergence of raised lesions in later life is to a great extent foreshadowed by youthful fatty streaks. As pointed out by McGill 1$4there are certain discrepancies in this association which deserve emphasis. Generally speaking, young Negro males from several populations developed considerably more extensive aortic fatty streaks than non-Negro males. In this microscopic study we found also that Negroes had excessive lipid content in aortic fatty streaks. Furthermore, in this particular set of
192
C. RESTREPO,
cases it was also found that Guatemalans
had excessive amounts
R. E. TRACY
of lipid in aortic fatty
streaks in relation to the low tendency to develop raised lesions at older ages. These discrepancies suggest that some populations (i.e., New Orleans Negro, Durban Bantu and Guatemala)
have fatty streaks which do not progress or progress
raised lesions as compared to other populations. The leukocytic content of fatty streaks was found Geer et al.j, to show a close association Nevertheless,
as pointed
out by Geer
in this study, and in that of
with the later emergence et al., and
more slowly into
confirmed
of raised lesions.
in the present
study,
“Cellular infiltration occurs only when intimal lipid is moderate or abundant, but intimal lipid may be abundant without cellular infiltration.” The prevalence of necrosis in aortic fatty streaks extent of raised lesions.
is another
feature
showing
Thus, it appears that it is not solely the quantity that determines
its progression
since intimal
close association
with the
of intimal lipid in the fatty streak
lipid can be both extensive
(in terms of
gross extent of intimal surface involved with fatty streaks) and high in density per unit of arterial wall (as judged histologically) without progressing rapidly to fibrous plaques. Some other quality of the fatty streak (perhaps associated with cellular infiltration and foci of necrosis
as found
in this study) is responsible
for the generation
raised lesions. More precise identification or definition of this quality important missing link in the pathogenesis of atherosclerosis. The unidentified a predisposition ting monocytes
quality
to progress
associated
with leukocytic
would
cellular infiltration
of
fill an
and with
to raised lesions could reside in the lipid, in the infiltra-
and macrophages
that become foam cells, or in the mesenchymal
of the arterial wall. It is possible that under some environmental
conditions,
tissue
the lipid is
more injurious or more difficult to be removed by the monocytes. The monocytes and foam cells may, under some conditions, be less efficient in the ingestion and removal of lipid. Smooth muscle and connective tissue may, also under some conditions, react with a stronger proliferative response than under other conditions. There might also be humoral differences among these groups that would be responsible for the differences in leukocytic response. For example, the well established differences in prevalence of the various risk factors (particularly hyperlipidemia, hypertension, and cigarette smoking) might affect lipid influx, metabolism, and efflux or the efficiency with which the macrophages dispose of phagocytosed lipid. Some other findings also deserve some consideration. The rarity with which mural thrombus was found in aortic fatty streaks and the conspicuous absence of vascularization and hemorrhage tend to add weight against hypotheses of a major role for these processes in the early development of atherosclerotic lesionsgvrO. In addition, calcification does not seem to be related to the propensity to convert fatty streaks into fibrous plaques. The finding of an unexpectedly high frequency of calcification in Lima could be related to the well established fact that water hardness (and calcium content of the drinking water) is extremely high in this locationll. From our findings we conclude that (1) raised lesions in older ages are partially predicted by fatty streaks in the young, (2) some location-race groups do not develop
VARIATIONS
IN HUMAN
AORTIC
the degree of raised lesions young,
and (3) leukocytic
location-race
groups
FATTY
expected
infiltration
STREAKS
AMONG
from the extent
GEOGRAPHIC
of fatty streaking
and foci of necrosis
with the higher propensities
LOCATIONS
193
seen in the
in fatty streaks mark those
to develop
raised lesions.
ACKNOWLEDGEMENTS
We are indebted
to Drs. Henry C. McGill,
Jr., Jack P. Strong
and Douglas
A.
Eggen for constructive criticism and encouragement during the development of the study and preparation of the manuscript. The valuable assistance of Mrs. Martha T. McMurry and Mrs. Vivian T. Toca regarding acknowledged.
the statistical
analyses
is also gratefully
REFERENCES
I MCGILL, JR., H. C., The lesion, Proccvdings of‘ /he Thitd Infernutioml Symposium on Athrro.sc/wosis, West Berlin, October 1973, In press. 2 MCGILL, JR., H. C., ARIAS-STELLA, J., CARBONELL, L. M.. CORREA, P., DEVEYRA, JR., E. A., DONOSO, S., EGGEN, D. A., GALINDO, L., GUZMAN, M. A., LKHTENBERGER, E.. LOKEN, A. C., MCGARRY, P. A., MCMAHAN, C. A., MONTENEGRO, M. R., Moossu, J., PEREZ-TAMAYO, R., RESTREPO, C., ROBERTSON, W. B., SALAS, J., SOLBERG, L. A., STKONC, J. P., TEJADA, C. AND WAINWRIGHT, J., General findings of the international atherosclerosis project, Lab. /nvr.st., 18 (1968) 498. 3 TEJADA, C., STRONG, J. P., MONTENEGRO, M. R., RESTREPO, C. AND SOLBERG, L. A., Distribution of coronary and aortic atherosclerosis by geographic location, race and sex, Lub. Inwsr., 18 ( 1968) 509. 4 MCGILL, JR., H. C., Fatty streaks in the coronary arteries and aorta, Lab. Imvsf., 18 (1968) 560. 5 GEER, J. C., MCGILL, JR., H. C., ROBERTSON, W. B. AND STRONG, J. P., Histologic characteristics of coronary artery fatty streaks, Lob. Invest., 18 (1967) 565. 6 GUZMAN, M. D., MCMAHAN, C. A., MCGILL, JR., H. C., STRohti, J. P., TEJADA, C., RESTREPO, C., EGGEN, D. A., ROBERTSON, W. B. AND SOLBERG, L. A., Selected methodologic aspects of the international atherosclerosis project, Lab. Invrst., 18 (1968) 479. 7 SNEDECOR, G. W. AND COCHRAN, W. G., Statistic’ul Mefhocls, 6th edition, The Iowa State University Press, Ames, Iowa, 1967, pp. 166-170. 8 MCMAHAN, C. A., Autopsied cases by age, sex and “race”, Lab. Invest., 18 (1968) 468. 9 HAUST, M. D., The morphogenesis and fate of potential and early atherosclerotic lesions, Hutmt~ f’urhology. 2 ( I97 I ) I IO HAUST, M. D. AND MORE, R. H., Development of modern theories on the pathogenesis of atherosclerosis. In: R. W. WISSLER AND J. C. GEER (Eds.), Puthogu7esi.r oj’Afhuosclwosi.q The Williams and Wilkins Co., Baltimore, Md., 1972, pp. I-19. 1 I STRONG, J. P., CORREA, P. AND SOLBERG, L. A.. Water hardness and atherosclerosis, Lab. Imwr., I8 ( 1968) 620.
